Enzyme activated self-immolative n-substituted nitrogen mustard prodrugs

ABSTRACT

This invention pertains to certain enzyme (CPG2) activated self-immolative nitrogen mustard prodrugs, which are useful in enzyme prodrug therapy (EPT), such as ADEPT and GDEPT, for the treatment of proliferative conditions, such as cancer, and which have the following formula: 
                         
wherein: R N  is independently C 1-7 alkyl; X 1  is independently —I, —Br, or —Cl; X 2  is independently —I, —Br, or —Cl; the group —N(CH 2 CH 2 X 1 )(CH 2 CH 2 X 2 ) is independently attached at the 2-position or at the 4-position; each R G  is independently —H or an ester substituent; n is independently an integer from 0 to 4; each R P , if present, is independently a phenyl substituent; m is independently an integer from 0 to 4; each R M , if present, is independently a mustard substituent; and pharmaceutically acceptable salts, solvates, amides, and esters thereof. The present invention also pertains to pharmaceutical compositions comprising such compounds; such compounds and compositions for use in methods of treatment of the human or animal body by therapy; the use of such compounds and compositions for the manufacture of medicaments for the treatment of proliferative conditions; and the like.

This application is the US national phase of international applicationPCT/GB2003/003736 filed 1 Sep. 2003 which designated the U.S. and claimsbenefit of GB 0220319.8, dated 2 Sep. 2002, the entire content of whichis hereby incorporated by reference.

TECHNICAL FIELD

This invention pertains generally to the field of chemotherapy, and morespecifically to certain enzyme (CPG2) activated self-immolative nitrogenmustard prodrugs which are useful in enzyme prodrug therapy (EPT), suchas ADEPT and GDEPT, for the treatment of proliferative conditions, suchas cancer. The present invention also pertains to pharmaceuticalcompositions comprising such compounds; such compounds and compositionsfor use in methods of treatment of the human or animal body by therapy;the use of such compounds and compositions for the manufacture ofmedicaments for the treatment of proliferative conditions; and the like.

BACKGROUND

Throughout this specification, including any claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps, butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and any appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

Chemotherapy

In general, cancer treatment by chemotherapy is limited by the need todeliver a high concentration of the anti-cancer drug selectively to themalignant cells. As a consequence, many methods for the efficient andselective delivery of a drug to the targeted malignant cells have beendeveloped.

Many such methods employ prodrugs, which may be described, generally, aspharmacologically inactive (or relatively inactive) chemical derivativesof a drug molecule that require a transformation within the body inorder to release the active drug.

In one approach, known as enzyme prodrug therapy (EPT), thetransformation is effected by a particular enzyme, for example,carboxypeptidase G2 (CPG2) or nitroreductase (NR). Examples of suchtherapies include antibody directed enzyme prodrug therapy (ADEPT) andgene directed enzyme prodrug therapy (GDEPT), briefly described below.Other enzyme prodrug therapies include ligand-directed enzyme prodrugtherapy (LIDEPT) (see, e.g., Springer and Marais, 1997) and bacteriadirected enzyme prodrug therapy (BDEPT) (see, e.g., Satchi and Duncan,1998). See also, for example, Kirn, 2000.

ADEPT

The ADEPT approach separates the targeting from the cytotoxic functionsin a two-step treatment. The selective component is an antibody (Ab)within an enzyme conjugate. The Ab binds antigen preferentiallyexpressed on the surface of tumour cells. In the first step, theAb-enzyme conjugate is administered and time is allowed for it toaccumulate at the tumour and to clear from blood and normal tissues. Inthe second step, a non-toxic prodrug is administered that is convertedspecifically by the enzyme at the tumour into a low molecular weighttoxic drug. The interstitial tumour transport of these low molecularweight cytotoxic agents thus generated is more favoured than those oflarge immuno-conjugates such as immunotoxins. This allows greater tumouraccess for the toxic component. An amplification feature is inherent inADEPT whereby one Ab-enzyme conjugate molecule can catalyse theconversion of many molecules of the prodrug into the cytotoxic drug,enabling higher concentrations of drug at the tumour than one-step Abdelivery systems. Another important factor is the by-stander effect,which effects killing of surrounding tumour cells even though they donot express tumour antigen or do not bind Ab-enzyme conjugate. The maindrawback currently remains the immunogenicity of the Ab-enzymeconjugates which precludes the administration of repeated doses of theconjugate.

A number of papers review in detail the main features of ADEPT systems,including: Senter et al., 1993; Bagshawe et al., 1994; Deonarain andEpenetos, 1994; Jungheim and Shepherd, 1994; Niculescu-Duvaz andSpringer, 1995, 1996; Springer and Niculescu-Duvaz, 1995; Springer etal., 1995a; Hay and Denny, 1996; Melton and Sherwood, 1996.

GDEPT

Gene therapy for cancer may be defined broadly as a genetic technologyaimed at modifying either malignant or non-malignant cells fortherapeutic gain. “Suicide” gene therapy approaches include GDEPT andVDEPT (virally directed enzyme prodrug therapy) (see, for example, Huberet al., 1995), the only difference between these approaches being thatthe former involves both viral and non-viral vectors.

Like ADEPT, GDEPT is a two-step treatment for tumours. Foreign enzymesare delivered to, and expressed in, target cells where they can activatesubsequently administered non-toxic prodrugs to form active drugs. Inthe first step, a gene expressing the foreign enzyme is delivered. Inthe second step, a prodrug is administered that can be activated to forma toxic drug by the enzyme that has been expressed in the tumour. Theforeign enzyme gene should be expressed exclusively, or with arelatively high ratio, in tumour cells compared with normal tissues andblood, and should achieve a sufficient concentration for clinicalbenefit. After gene delivery, prodrug administration must be delayed topermit protein expression in the targeted cells. The catalytic activityof the expressed protein should be sufficient for activation of theprodrug. Since expression of the foreign enzymes will not occur in allcells of a targeted tumour in vivo, a bystander cytotoxic effect isbeneficial, whereby the prodrug is cleaved to an active drug that killsnot only tumour cells but also neighbouring non-expressing tumour cells.This means that expression in less than 100% of tumour cells can stillresult in killing of all tumour cells. The foreign enzyme is usuallyexpressed intracellularly, but by expressing the activating enzymetethered to the outer cell surface of mammalian cells, potentialadvantages for GDEPT prodrug design are realized. The potentialadvantages of extracellular expression are twofold. Firstly, it shouldgive an improved by-stander effect because the drug will be generated inthe interstitial spaces within the tumour, rather than inside as with anintracellularly expressed activating enzyme. Secondly, the prodrugcannot enter cells to become activated and therefore non-cell-permeableprodrugs can be used. Thus, prodrugs which release drugs withintracellular targets may be rendered non-toxic by preventing theirentry into cells. Upon activation, a potent and cell-permeable activemoiety is released. This has already been demonstrated to be beneficialfor prodrug-impermeable tumour cells (Marais et al., 1997). However, thepotential for increased toxicity due to the diffusion of the active drugaway from the tumour is a potential disadvantage, although this couldalso happen to active drugs from an intracellularly expressed enzyme.

A number of recent reviews cover the GDEPT approach, including: Zhang etal., 1995; Niculescu-Duvaz and Springer, 1997; Roth and Cristiano, 1997;Denny and Wilson, 1998; Encell and Loeb, 1998; Niculescu-Duvaz et al.,1998a, 1999; Springer and Niculescu-Duvaz, 1999a. Additional aspects aredescribed in Springer and Marais, 1996a, 1996b.

Carboxypeptidase G2 (CPG2)

Peptidases are a class of enzymes (E) which act upon a substrate tocleave an amide linkage (—NH—C(═O)—) to give, usually, amino (—NH₂) andcarboxylic acid (—C(═O)OH) products.

One peptidase of particular interest is carboxypeptidase G2 (referred toherein as “CPG2”). CPG2 is a bacterial enzyme isolated from PseudomonasR16 (Sherwood et al., 1985). It is a zinc-dependent metallo-proteinasewhich exists as a homodimer molecule (2×41,800 Da) containing two Zn²⁺ions in each monomeric unit (Minton et al., 1984). The enzyme belongs tothe group of calcium-binding zinc-endopeptidases from bacteria whichcontain thermolysin and other neutral peptidases from Bacillus subtilisand Aeromonas proteolytica (Matthews, 1988; Roswell et al., 1997).

CPG2 was first proposed by Bagshawe et al., 1988, and catalyses thescission of amidic (Springer et at., 1990a), urethanic or ureidic(Springer et al., 1995b; Dowell et al., 1996), bonds between a benzenenucleus and L-glutamic acid.

A preferred substrate for CPG2 is an L-glutamic acid group, linked to anaromatic ring via an amidic, carbamic, or ureidic linkage.

However, glutamic acid analogs are also acceptable substrates. Forexample, L-glutamic acid modified at the γ-carbon (e.g., with an amide,—CONH₂, instead of an acid, —COOH) also serves as a suitable substratefor CPG2.

CPG2 is also tolerant as to whether the amide group is naked, or is partof a larger linkage, for example, a carbamate or a urea linkage.

For these compounds, CPG2 yields CO₂, L-glutamic acid, and R-ZH, whereinwhen Z is —O— (carbamates), R-ZH is a hydroxyl compound, R—OH, and whenZ is —NH— (ureas), R-ZH is an amino compound, R—NH₂, where R ispreferably an aromatic group.

CPG2 Activated Self-Immolative Prodrugs

A “self-immolative prodrug” can be defined as a compound which,following an activation process, generates an unstable intermediate thatreleases the active drug in a number of subsequent steps.

Typically: (i) the activation process is of an enzymatic nature and isdistinct from the extrusion step; (ii) the drug is generated by anextrusion process, following the fragmentation of the prodrug; (iii) thesite of activation will normally be separated from the site ofextrusion.

Potential advantages of self-immolative prodrugs include: thepossibility of altering the lipophilicity of the prodrugs with minimaleffect on the activation kinetics; the improvement of unfavourablekinetics of activation due to unsuitable electronic or steric features;the range of drugs which can be converted to prodrugs is greatlyextended and is unrestricted by the structural substrate requirementsfor a given enzyme.

In one class of CPG2 activated self-immolative prodrugs, shown below,the L-glutamic acid and the active drug are separated by a 4-hydroxy(where Z is —O—) or 4amino where Z is —NH—) substituted benzylic spacer.

The activation of these prodrugs involves two steps:

-   -   (i) the cleavage of the oxycarbonyl- or carbamoyl-L-glutamyl        linkage by CPG2, followed by the spontaneous decomposition of        the carbonic or carbamic acid thus formed with loss of CO₂;    -   (ii) the fragmentation of the self-immolative intermediate by a        1,6-elimination mechanism (Wakselman, 1983), releasing a        carbonic or carbamic acid which upon loss of CO₂ generates an        active drug (HNR₂).

In this way, the prodrug, upon self-immolation, releases an amine drug(and CO₂) from a carbamate linkage. Other classes includes compoundswhich, upon self-immolation, release an aryl alcohol from an aryl ether;an aryl carboxylic acid from an aryl ester; an aryl alcohol (and CO₂)from an aryl carbonate; and the like. Similar self-immolative prodrugsare described, for example, in Springer et al, 1995c, 1995d.

Nitrogen Mustards

Nitrogen mustards are related to sulfur mustard, (ClCH₂CH₂)₂S, the“mustard gas” used during the First World War. Nitrogen mustards havethe general formula (ClCH₂CH₂)₂NR. In vivo, each 2-chloroethylside-chain undergoes an intramolecular cyclisation with the release of achloride ion. The resulting highly reactive ethylene immonium derivativecan interact with DNA and other molecules, for example, as an alkylatingand/or crosslinking agent. Nitrogen mustards are useful, for example, inthe treatment of proliferative conditions, such as cancer.

Nitrogen mustard analogues, in which the chloro group is replaced byother groups, such as other halogens (e.g., bromo, iodo) and other goodleaving groups (e.g., sulfonates, such as mesyloxy, —OSO₂Me) are alsoknown, and are included in the class denoted “nitrogen mustards.”

Nitrogen mustards may conveniently be grouped according to the group R.For example, two groups are phenolic nitrogen mustards and anilinicnitrogen mustards.

CPG2 Activated Nitrogen Mustard Prodrugs

The EPT approach has been applied to nitrogen mustard drugs. Forexample, in one approach, CPG2 acts upon the prodrug to yield a drug,R-ZH, which is a phenolic (Z is —O—) or anilinic (Z is —NH—) nitrogenmustard compound.

Various nitrogen mustard prodrugs are described, for example, inSpringer 1990b, 1991, 1994, 2000, and 2002.

CPG2 Activated Nitrogen Mustard Self-Immolative Prodrugs

The CPG2 activated self-immolative prodrug approach, discussed above,has also been applied to nitrogen mustards. In one approach, the drug,NHR₂, is an anilinic nitrogen mustard compound. The prodrug is activatedby CPG2, undergoes self-immolation, and releases the anilinic nitrogenmustard.

Springer et al, 1996, describe a number of nitrogen mustard prodrugs,including compounds of the following structure (see, for example,compounds 19 and 20 on pages 22 and 23 and in FIG. 3, therein).

In each case, the nitrogen atom (indicated by the arrow, above) of thecarbamate group which is between the benzyl group of the self-immolativecore and the phenyl group of the nitrogen mustard is unsubstituted, thatis, the carbamate group is —O—C(═O)—NH—. This nitrogen atom, identifiedas Z¹ therein, is soley described as —O— or —NH— (see page 3, line 5;page 6, line 24; page 7, line 1, page 7, line 7; page 11, line 1; andpage 15 line 6, therein). Nowhere in this document is there provided anyteaching or suggestion whatsoever that, as an alternative, the nitrogenatom of this carbamate group might be substituted.

The inventors have discovered that, surprisingly and unexpectedly,corresponding compounds, in which the nitrogen atom is substitued, forexample, with a C₁₋₇alkyl group, offer one or more pharmacologicaladvantages, including but not limited to: (a) improved activity; (b)improved selectivity (e.g., against tumour cells versus normal cells);(c) reduction in required dosage amounts; (d) reduction in requiredfrequency of administration; (e) reduced intensity of undesiredside-effects; (f) fewer undesired side-effects.

SUMMARY OF THE INVENTION

One aspect of the invention pertains to compounds (prodrugs), asdescribed herein.

Another aspect of the present invention pertains to compounds (prodrugs)which (a) regulate (e.g., inhibit) cell proliferation; (b) inhibit cellcycle progression; (c) promote apoptosis; or (d) a combination of one ormore of these.

Another aspect of the invention pertains to compounds (prodrugs), asdescribed herein, which are anticancer agents.

Another aspect of the invention pertains to compounds (prodrugs), asdescribed herein, which are antiproliferative agents.

Another aspect of the present invention pertains to a compositioncomprising a compound, as described herein, and a carrier.

Another aspect of the present invention pertains to a compositioncomprising a compound, as described herein, and a pharmaceuticallyacceptable carrier.

Another aspect of the present invention pertains to a method of (a)regulating (e.g., inhibiting) proliferation of a cell; (b) inhibitingcell cycle progression of a cell; (c) promoting apoptosis of a cell; or(d) a combination of one or more of these, in vitro or in vivo,comprising contacting the cell with an effective amount of a compound(prodrug), as described herein.

Another aspect of the present invention pertains to a method ofregulating (e.g., inhibiting) proliferation of a cell, in vitro or invivo, comprising contacting the cell with an effective amount of acompound (prodrug), as described herein.

Another aspect of the present invention pertains to a method oftreatment, for example, of cancer, a proliferative condition, or othercondition as described herein, comprising administering to a subject inneed of treatment a therapeutically-effective amount of a compound(prodrug), as described herein, preferably in the form of apharmaceutical composition.

Another aspect of the present invention pertains to a compound (prodrug)for use in a method of treatment of the human or animal body by therapy,for example, in the treatment of cancer, a proliferative condition, orother condition as described herein.

Another aspect of the present invention pertains to the use of acompound (prodrug) for the manufacture of a medicament, for example, forthe treatment of cancer, a proliferative condition, or other conditionas described herein.

Another aspect of the present invention pertains to a method formanufacturing a medicament intended for therapeutic application, forexample, for the treatment of cancer, a proliferative condition, orother condition as described herein, characterised in that a compound(prodrug), as described herein, is used.

Another aspect of the invention pertains to a kit comprising (a) thecompound (prodrug), preferably provided in a suitable container and/orwith suitable packaging; and (b) instructions for use, for example,written instructions on how to administer the compound (prodrug), etc.

Another aspect of the present invention pertains to a method of enzymeprodrug therapy (EPT) which employs a compound (prodrug), as describedherein, and a carboxypeptidase enzyme, as described herein.

Another aspect of the present invention pertains to a method of antibodydirected enzyme prodrug therapy (ADEPT) which employs a compound(prodrug), as described herein, and a carboxypeptidase enzyme, asdescribed herein.

Another aspect of the present invention pertains to a two componentsystem (comprising two components for use in association with oneanother), comprising: (a) a prodrug, as described herein; and (b) anantibody or fragment thereof conjugated or fused to a carboxypeptidaseenzyme, as described herein.

Another aspect of the present invention pertains to a method of genedirected enzyme prodrug therapy (GDEPT) which employs a compound(prodrug), as described herein, and a carboxypeptidase enzyme, asdescribed herein.

Another aspect of the present invention pertains to a two componentsystem (comprising two components for use in association with oneanother), comprising: (a) a prodrug, as described herein; and (b) anucleic acid encoding (e.g., as part of a vector capable of expressing)a carboxypeptidase enzyme, as described herein.

Another aspect of the present invention pertains to compounds (e.g.,intermediates, prodrugs, etc.) obtainable by a method of synthesis asdescribed herein, or a method comprising a method of synthesis asdescribed herein.

Another aspect of the present invention pertains to compounds (e.g.,intermediates, prodrugs, etc.) obtained by a method of synthesis asdescribed herein, or a method comprising a method of synthesis asdescribed herein.

Another aspect of the present invention pertains to novel intermediates,as described herein (including, for example, compounds 7 and 8 in Scheme2 and compounds 20, 21, 22, and 23 in Scheme 11), which are suitable foruse in the methods of synthesis described herein.

Another aspect of the present invention pertains to the use of suchnovel intermediates, as described herein, in the methods of synthesisdescribed herein.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Compounds

One aspect of the present invention pertains to compounds of theformula:

wherein:

-   -   R^(N) is independently C₁₋₇alkyl;    -   X¹ is independently —I, —Br, or —Cl;    -   X² is independently —I, —Br, or —Cl;    -   the group —N(CH₂CH₂X¹)(CH₂CH₂X²) is independently attached at        the 2-position or at the 4-position;    -   each R^(G) is independently —H or an ester substituent (R^(E));    -   n is independently an integer from 0 to 4;    -   each R^(P), if present, is independently a phenyl substituent;    -   m is independently an integer from 0 to 4;    -   each R^(M), if present, is independently a mustard substituent;    -   and pharmaceutically acceptable salts, solvates, amides, and        esters thereof.        Nitrogen Substituent, R^(N)

The nitrogen substituent, R^(N), is independently C₁₋₇alkyl.

In one embodiment, R^(N), is independently aliphatic C₁₋₇alkyl.

In one embodiment, R^(N), is independently unsubstituted C₁₋₇alkyl.

In one embodiment, R^(N), is independently unsubstituted aliphaticC₁₋₇alkyl.

In one embodiment, R^(N) is independently C₁₋₄alkyl.

In one embodiment, R^(N) is independently aliphatic C₁₋₄alkyl.

In one embodiment, R^(N) is independently unsubstituted C₁₋₄alkyl.

In one embodiment, R^(N) is independently unsubstituted aliphaticC₁₋₄alkyl.

In one embodiment, R^(N) is independently -Me, -Et, -nPr, -iPr, -allyl,-nBu, -sBu, -iBu, or -tBu.

In one embodiment, R^(N) is independently -Me or -Et.

In one embodiment, R^(N) is independently -Me.

Mustard Substituents, X¹ and X²

Each of the mustard substituents, X¹ and X², is independently —I, —Br,or —Cl.

In one embodiment, each of X¹ and X² is independently —I, —Br, or —Cl;and both of X¹ and X², are the same.

In one embodiment, each of X¹ and X² is independently —I or —Br.

In one embodiment, each of X¹ and X² is independently —I or —Br; andboth of X¹ and X² are the same.

In one embodiment, each of X: and X² is independently —I.

In one embodiment, each of X¹ and X² is independently —Br.

In one embodiment, each of X¹ and X² is independently —Cl.

Position of the Nitrogen Mustard Group

The nitrogen mustard group, —N(CH₂CH₂X)₂, is independently attached atthe 2-position (“ortho”) or at the 4-position (“para”).

In one embodiment, the nitrogen mustard group, —N(CH₂CH₂X¹)(CH₂CH₂X²),is independently attached at the 2-position (“ortho”).

In one embodiment, the nitrogen mustard group, —N(CH₂CH₂X¹)(CH₂CH₂X²),is independently attached at the 4-position (“para”).

Phenyl Substituents, R^(P)

The phenylene group of the self-immolative core optionally bears phenylsubstituents, R^(P):

In one embodiment, n is 0, 1, 2, 3, or 4.

In one embodiment, n is 0, 1, 2, or 3.

In one embodiment, n is 0, 1, or 2.

In one embodiment, n is 0 or 1.

In one embodiment, n is 1, 2, 3, or 4.

In one embodiment, n is 1, 2, or 3.

In one embodiment, n is 1, or 2.

In one embodiment, n is 4.

In one embodiment, n is 3.

In one embodiment, n is 2.

In one embodiment, n is 1.

In one embodiment, n is 0.

In one embodiment, each R^(P), if present, is independently halo,C₁₋₄alkyl, nitro, or cyano.

In one embodiment, each R^(P), if present, is independently —F, —Cl,—Br, —I, -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu, —NO₂, or —CN.

In one embodiment, each R^(P), if present, is independently —F, —Cl,—Br, or —I.

In one embodiment, each R^(P), if present, is independently —F, —Cl or—Br.

In one embodiment, each R^(P), if present, is independently —F or —Cl.

In one embodiment, each R^(P), if present, is independently —F or —Br.

In one embodiment, each R^(P), if present, is independently —F.

In one embodiment, the phenylene group has the following formula:

wherein each of R^(P2), R^(P3), R^(P5), and R^(P6) is independently —Hor an phenyl substituent.

In one embodiment, each of R^(P2), R^(P3), R^(P5), and R^(P6) isindependently —H or a phenyl substituent, as described above for R^(P).

In one embodiment, each of R^(P2) and R^(P6) is —H (and each of R^(P3)and R^(P5) is other than —H, for example, as described above for R^(P)):

In one embodiment, each of R^(P2), R^(P5), and R^(P6) is —H (and R^(P3)is other than —H, for example, as described above for R^(P)):

In one embodiment, each of R^(P2), R^(P3), R^(P5),and R^(P6) is —H.

Mustard Substituents, R^(M)

The phenylene group, to which the nitrogen mustard group is attached,optionally also bears mustard substituents, R^(M):

In one embodiment, m is 0, 1, 2, 3, or 4.

In one embodiment, m is 0, 1, 2, or 3.

In one embodiment, m is 0, 1, or 2.

In one embodiment, m is 0 or 1.

In one embodiment, m is 1, 2, 3, or 4.

In one embodiment, m is 1, 2, or 3.

In one embodiment, m is 1, or 2.

In one embodiment, m is 4.

In one embodiment, m is 3.

In one embodiment, m is 2.

In one embodiment, m is 1.

In one embodiment, m is 0:

In one embodiment, each R^(M), if present, is independently selectedfrom:

-   -   C₁₋₄alkyl (including, e.g., C₁₋₄haloalkyl);    -   C₁₋₄alkoxy (including, e.g., C₁₋₄haloalkoxy);    -   amino (including, e.g., di-C₁₋₄alkyl amino);    -   halo;    -   C₁₋₄alkylthio;    -   acyl (e.g., C₁₋₇alkyl-acyloxy, C₅₋₆aryl-acyloxy);    -   ester;    -   amido;    -   cyano;    -   nitro; and,    -   C₅₋₆aryl.

In one embodiment, each R^(M), if present, is independently selectedfrom:

-   -   -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu;    -   —CF₃, —CH₂F, —CH₂CF₃, —CH₂CH₂F; —CF₂CF₃;    -   —OMe, —OEt, —O-nPr, —O-iPr, —O-nBu, —O-sBu, —O-iBu, —O-tBu;    -   —OCF₃, —OCH₂F, —OCH₂CF₃, —OCH₂CH₂F; —OCF₂CF₃;    -   —NH₂, —NMe₂, —NEt₂, —N(nPr)₂, —N(iPr)₂,    -   —F, —Cl, —Br, —I;    -   —SMe, —SEt;    -   —C(═O)Me;    -   —C(═O)OMe, —C(═O)OEt;    -   —CONH₂, —CONHMe;    -   —CN;    -   —NO₂; and,    -   -Ph.

In one embodiment, each R^(M), if present, is independently selectedfrom:

-   -   C₁₋₄alkyl (including, e.g., C₁₋₄haloalkyl);    -   C₁₋₄alkoxy (including, e.g., C₁₋₄haloalkoxy); and,    -   amino (including, e.g., di-C₁₋₄-alkyl amino).

In one embodiment, each R^(M), if present, is independently selectedfrom:

-   -   -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu;    -   —CF₃, —CH₂F, —CH₂CF₃, —CH₂CH₂F; —CF₂CF₃;    -   —OMe, —OEt, —O-nPr, —O-iPr, —O-nBu, —O-sBu, —O-iBu, —O-tBu;    -   —OCF₃, —OCH₂F, —OCH₂CF₃, —OCH₂CH₂F; —OCF₂CF₃;    -   —NH₂, —NMe₂, —NEt₂, —N(nPr)₂, and —N(iPr)₂,

In one embodiment, each R^(M), if present, is independently selectedfrom:

-   -   -Me, -Et, —CF₃, —OMe, —OEt, —NH₂, and —NMe₂.

In general, mustard substituents, R^(M), which are electron-withdrawingand which decrease the chemical reactivity of the resulting nitrogenmustard are generally less preferred.

Glutamic Acid Ester Substituents, R^(G)

Each of the glutamic acid groups, R^(G), is independently —H or an estersubstiuent (R^(E)).

In one embodiment, each of the glutamic acid groups, R^(G), isindependently —H.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently an ester substituent (R^(E)).

In one embodiment, each of the glutamic acid groups, R^(G), isindependently —H, unsubstituted C₁₋₇alkyl, substituted C₁₋₇alkyl, orsilyl.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently —H, unsubstituted C₁₋₇alkyl, or substituted C₁₋₇alkyl.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently —H or unsubstituted C₁₋₇alkyl.

In one embodiment, the unsubstituted C₁₋₇alkyl group is independentlyunsubstituted C₁₋₄alkyl.

In one embodiment, the unsubstituted C₁₋₇alkyl group is independently:-Me, -Et, -nPr, -iPr, -allyl, -nBu, -sBu, -iBu, or -tBu.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁₋₇alkyl substituted with one or more groups selected from optionallysubstituted C₅₋₂₀aryl, C₁₋₇alkoxy, C₁₋₇alkylthio, and acyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁₋₄alkyl substituted with one or more groups selected from optionallysubstituted C₅₋₂₀aryl, C₁₋₇alkoxy, C₁₋₇alkylthio, and acyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁alkyl substituted with one or more groups selected from optionallysubstituted C₅₋₂₀aryl, C₁₋₇alkoxy, C₁₋₇alkylthio, and acyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁₋₇alkyl substituted with one or more groups selected from optionallysubstituted C₅₋₆aryl, C₁₋₄alkoxy, C₁₋₄alkylthio, C₁₋₄alkyl-acyloxy,C₅₋₆aryl-acyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁₋₄alkyl substituted with one or more groups selected from optionallysubstituted C₅₋₆aryl, C₁₋₄alkoxy, C₁₋₄alkylthio, C₁₋₄alkyl-acyloxy,C₅₋₆aryl-acyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁alkyl substituted with one or more groups selected from optionallysubstituted C₅₋₆aryl, C₁₋₄alkoxy, C₁₋₄alkylthio, C₁₋₄alkyl-acyloxy,C₅₋₆aryl-acyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁₋₇alkyl substituted with one or more groups selected from optionallysubstituted phenyl (e.g., methoxyphenyl, nitrophenyl), methoxy,methylthio, acetoxy, and benzoyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁₋₄alkyl substituted with one or more groups selected from optionallysubstituted phenyl (e.g., methoxyphenyl, nitrophenyl), methoxy,methylthio, acetoxy, and benzoyloxy.

In one embodiment, the substituted C₁₋₇alkyl group is independentlyC₁alkyl substituted with one or more groups selected from optionallysubstituted phenyl (e.g., methoxyphenyl, nitrophenyl), methoxy,methylthio, acetoxy, and benzoyloxy.

In one embodiment, the silyl group is independently —SiR^(S) ₃, whereineach R^(S) is independently —H or C₁₋₄alkyl.

In one embodiment, the silyl group is independently —Si(Me)₃, —Si(Et)₃,—Si(iPr)₃, —Si(tBu)(CH₃)₂, or —Si(tBu)₃.

In one embodiment, the silyl group is independently —Si(iPr)₃.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently (1) t-butyl, (2) allyl, (3) tri-isopropylsilyl, (4)acetoxymethyl, (5) methoxymethyl, (6) methylthiomethyl, (7)p-methoxyphenylmethyl, (8) bis(o-nitrophenyl)methyl, (9) benzyl, or (10)diphenylmethyl.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently (1) t-butyl, (2) allyl, (3) tri-isopropylsilyl, (4)acetoxymethyl, or (5) methoxymethyl.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently (1) t-butyl, (2) allyl, or (3) tri-isopropylsilyl.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently (1) t-butyl or (2) allyl.

In one embodiment, each of the glutamic acid groups, R^(G), isindependently (1) allyl.

Glutamic Acid Group Configuration

That part of the compound having the following formula is referred toherein as “the glutamic acid group”:

The carbon atom marked with an asterisk (*) is a chiral centre, and maybe in an R or an S configuration.

In one embodiment, the carbon atom marked with an asterisk (*) isindependently in an S configuration, and the glutamic acid group is anL-glutamic acid group, of the following formula:

Certain Preferred Embodiments

(1) In one embodiment:

(A1) R^(N) is independently C₁₋₄alkyl; and,

(B1) each X is independently —Cl, —Br or —I.

(2) In one embodiment:

(A2) R^(N) is independently -Et or -Me; and,

(B1) each X is independently —Cl, —Br or —I.

(3) In one embodiment:

(A3) R^(N) is independently -Me; and,

(B1) each X is independently —Cl, —Br or —I.

(4) In one embodiment:

(A1) R^(N) is independently C₁₋₄alkyl; and,

(B2) each X is independently —Br or —I.

(5) In one embodiment:

(A2) R^(N) is independently -Et or -Me; and,

(B2) each X is independently —Br or —I.

(6) In one embodiment:

(A3) R^(N) is independently -Me; and,

(B2) each X is independently —Br or —I.

(7) In one embodiment:

(A1) R^(N) is independently C₁₋₄alkyl; and,

(B3) each X is independently —I.

(8) In one embodiment:

(A2) R^(N) is independently -Et or -Me; and,

(B3) each X is independently —I.

(9) In one embodiment:

(A3) R^(N) is independently -Me; and,

(B3) each X is independently —I.

(10) to (18): Each of the above embodiments (1) to (9), whereinfurthermore:

(C1) the group —N(CH₂CH₂X)₂ is independently attached at the 4-position(“para”).

(19) to (27): Each of the above embodiments (10) to (18), whereinfurthermore:

(D1) n is independently 0.

(28) to (36): Each of the above embodiments (19) to (27), whereinfurthermore:

(E1) m is independently 0.

(37) to (45): Each of the above embodiments (28) to (36), whereinfurthermore:

(F1) R^(G) is independently —H.

(46) to (54): Each of the above embodiments (28) to (36), whereinfurthermore:

(F2) R^(G) is independently C₁₋₆alkyl.

Specific Embodiments

In one embodiment, the compound is selected from the followingcompounds, and pharmaceutically acceptable salts, solvates, amides, andesters thereof:

In one embodiment, the compound is selected from P-1, P-2, and P-3, andpharmaceutically acceptable salts, solvates, amides, and esters thereof.

In one embodiment, the compound is selected from P-1 andpharmaceutically acceptable salts, solvates, amides, and esters thereof.

In one embodiment, the compound is selected from P-2 andpharmaceutically acceptable salts, solvates, amides, and esters thereof.

In one embodiment, the compound is selected from P-3 andpharmaceutically acceptable salts, solvates, amides, and esters thereof.

Chemical Terms

The term “carbo,” “carbyl,” “hydrocarbon” and “hydrocarbyl,” as usedherein, pertain to compounds and/or groups which have only carbon andhydrogen atoms (but see “carbocyclic” below).

The term “hetero,” as used herein, pertains to compounds and/or groupswhich have at least one heteroatom, for example, multivalent heteroatoms(which are also suitable as ring heteroatoms) such as boron, silicon,nitrogen, phosphorus, oxygen, sulfur, and selenium (more commonlynitrogen, oxygen, and sulfur) and monovalent heteroatoms, such asfluorine, chlorine, bromine, and iodine.

The term “saturated,” as used herein, pertains to compounds and/orgroups which do not have any carbon-carbon double bonds or carbon-carbontriple bonds.

The term “unsaturated,” as used herein, pertains to compounds and/orgroups which have at least one carbon-carbon double bond orcarbon-carbon triple bond.

The term “aliphatic,” as used herein, pertains to compounds and/orgroups which are linear or branched, but not cyclic (also known as“acyclic” or “open-chain” groups).

The term “ring,” as used herein, pertains to a closed ring of from 3 to10 covalently linked atoms, more preferably 3 to 8 covalently linkedatoms, yet more preferably 5 to 6 covalently linked atoms. A ring may bean alicyclic ring or an aromatic ring. The term “alicyclic ring,” asused herein, pertains to a ring which is not an aromatic ring.

The term “carbocyclic ring,” as used herein, pertains to a ring whereinall of the ring atoms are carbon atoms.

The term “carboaromatic ring,” as used herein, pertains to an aromaticring wherein all of the ring atoms are carbon atoms.

The term “heterocyclic ring,” as used herein, pertains to a ring whereinat least one of the ring atoms is a multivalent ring heteroatom, forexample, nitrogen, phosphorus, silicon, oxygen, or sulfur, though morecommonly nitrogen, oxygen, or sulfur. Preferably, the heterocyclic ringhas from 1 to 4 heteroatoms.

The term “cyclic compound,” as used herein, pertains to a compound whichhas at least one ring. The term “cyclyl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof a cyclic compound.

Where a cyclic compound has two or more rings, they may be fused (e.g.,as in naphthalene), bridged (e.g., as in norbornane), spiro (e.g., as inspiro[3.3]heptane), or a combination thereof. Cyclic compounds with onering may be referred to as “monocyclic” or “mononuclear,” whereas cycliccompounds with two or more rings may be referred to as “polycyclic” or“polynuclear.”

The term “carbocyclic compound,” as used herein, pertains to a cycliccompound which has only carbocyclic ring(s).

The term “heterocyclic compound,” as used herein, pertains to a cycliccompound which has at least one heterocyclic ring.

The term “aromatic compound,” as used herein, pertains to a cycliccompound which has at least one aromatic ring.

The term “carboaromatic compound,” as used herein, pertains to a cycliccompound which has only carboaromatic ring(s).

The term “heteroaromatic compound,” as used herein, pertains to a cycliccompound which has at least one heteroaromatic ring.

The term “monodentate substituents,” as used herein, pertains tosubstituents which have one point of covalent attachment.

The term “monovalent monodentate substituents,” as used herein, pertainsto substituents which have one point of covalent attachment, via asingle bond. Examples of such substituents include halo, hydroxy, andalkyl.

The term “multivalent monodentate substituents,” as used herein,pertains to substituents which have one point of covalent attachment,but through a double bond or triple bond. Examples of such substituentsinclude oxo, imino, alkylidene, and alklidyne.

The term “bidentate substituents,” as used herein, pertains tosubstituents which have two points of covalent attachment, and which actas a linking group between two other moieties. Examples of suchsubstituents include alkylene and arylene.

Substituents

The phrase “optionally substituted,” as used herein, pertains to aparent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted,” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, appended to, or ifappropriate, fused to, a parent group. A wide variety of substituentsare well known, and methods for their formation and introduction into avariety of parent groups are also well known.

The substituents are described in more detail below.

Alkyl: The term “alkyl,” as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from a carbon atom of a hydrocarboncompound having from 1 to 20 carbon atoms (unless otherwise specified),which may be aliphatic or alicyclic, and which may be saturated,partially unsaturated, or fully unsaturated. Thus, the term “alkyl”includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussedbelow.

In this context, the prefixes (e.g., C₁₋₄, C₁₋₇, C₁₋₂₀, C₂₋₇, C₃₋₇,etc.) denote the number of carbon atoms, or range of number of carbonatoms. For example, the term “C₁₋₄alkyl,” as used herein, pertains to analkyl group having from 1 to 4 carbon atoms. Examples of groups of alkylgroups include C₁₋₄alkyl (“lower alkyl”), C₁₋₇alkyl, and C₁₋₂₀alkyl.

Examples of (unsubstituted) saturated alkyl groups include, but are notlimited to, methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl(C₅), hexyl (C₆), heptyl (C₇), octyl (C₈), nonyl (C₉), decyl (C₁₀),undecyl (C₁₁), dodecyl (C₁₂), tridecyl (C₁₃), tetradecyl (C₁₄),pentadecyl (C₁₅), and eicodecyl (C₂₀).

Examples of (unsubstituted) saturated linear alkyl groups include, butare not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl(C₄), n-pentyl (amyl) (C₅), n-hexyl (C₆), and n-heptyl (C₇).

Examples of (unsubstituted) saturated branched alkyl groups includeiso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄),iso-pentyl (C₅), and neo-pentyl (C₅).

Cycloalkyl: The term “cycloalkyl,” as used herein, pertains to an alkylgroup which is also a cyclyl group; that is, a monovalent moietyobtained by removing a hydrogen atom from an alicyclic ring atom of acyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 20ring atoms (unless otherwise specified). Preferably, each ring has from3 to 7 ring atoms.

Examples of (unsubstituted) saturated cylcoalkyl groups include, but arenot limited to, those derived from: cyclopropane (C₃), cyclobutane (C₄),cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C₇), norbornane (C₇),norpinane (C₇), norcarane (C₇), adamantane (C₁₀), and decalin(decahydronaphthalene) (C₁₀).

Examples of (substituted) saturated cycloalkyl groups, which are alsoreferred to herein as “alkyl-cycloalkyl” groups, include, but are notlimited to, methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl,dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl,methylcyclohexyl, and dimethylcyclohexyl, menthane, thujane, carane,pinane, bornane, norcarane, and camphene.

Examples of (substituted) unsaturated cyclic alkenyl groups, which arealso referred to herein as “alkyl-cycloalkenyl” groups, include, but arenot limited to, methylcyclopropenyl, dimethylcyclopropenyl,methylcyclobutenyl, dimethylcyclobutenyl, methylcyclopentenyl,dimethylcyclopentenyl, methylcyclohexenyl, and dimethylcyclohexenyl.

Examples of (substituted) cycloalkyl groups, with one or more otherrings fused to the parent cycloalkyl group, include, but are not limitedto, those derived from: indene (C₉), indan (e.g., 2,3-dihydro-1H-indene)(C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀), acenaphthene(C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene (C₁₅),aceanthrene (C₁₆). For example, 2H-inden-2-yl is a C₅cycloalkyl groupwith a substituent (phenyl) fused thereto.

Alkenyl: The term “alkenyl,” as used herein, pertains to an alkyl grouphaving one or more carbon-carbon double bonds. Examples of groups ofalkenyl groups include C₂₋₄alkenyl, C₂₋₇alkenyl, C₂₋₂₀alkenyl.

Examples of (unsubstituted) unsaturated alkenyl groups include, but arenot limited to, ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃),2-propenyl (allyl, —CH—CH═CH₂), isopropenyl (—C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

Examples of (unsubstituted) unsaturated cyclic alkenyl groups, which arealso referred to herein as “cycloalkenyl” groups, include, but are notlimited to, cyclopropenyl (C₃), cyclobutenyl (C₄), cyclopentenyl (C₅),and cyclohexenyl (C₆).

Alkynyl: The term “alkynyl,” as used herein, pertains to an alkyl grouphaving one or more carbon-carbon triple bonds. Examples of groups ofalkynyl groups include C₁₋₄alkynyl, C₂₋₇alkynyl, C₂₋₂₀alkynyl.

Examples of (unsubstituted) unsaturated alkynyl groups include, but arenot limited to, ethynyl (ethinyl, —C—CH) and 2-propynyl (propargyl,—CH₂—C≡CH).

Carbocyclyl: The term “carbocyclyl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof a carbocyclic compound, which moiety has from 3 to 20 ring atoms(unless otherwise specified). Preferably, each ring has from 3 to 7 ringatoms.

In this context, the prefixes (e.g., C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms. For example, theterm “C₅₋₆carbocyclyl,” as used herein, pertains to a carbocyclyl grouphaving 5 or 6 ring atoms. Examples of groups of carbocyclyl groupsinclude C₃₋₂₀carbocyclyl, C₃₋₁₀carbocyclyl, C₅₋₁₀carbocyclyl,C₃₋₇carbocyclyl, and C₅₋₇carbocyclyI.

Examples of carbocyclic groups include, but are not limited to, thosedescribed above as cycloalkyl groups; and those described below ascarboaryl groups.

Heterocyclyl: The term “heterocyclyl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof a heterocyclic compound, which moiety has from 3 to 20 ring atoms(unless otherwise specified), of which from 1 to 10 are ringheteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of whichfrom 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g., C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl,” as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.Examples of groups of heterocyclyl groups include C₃₋₂₀heterocyclyl,C₃₋₇heterocyclyl, C₅₋₇heterocyclyl, and C₅₋₆heterocyclyl.

Examples of heterocyclyl groups which are also heteroaryl groups aredescribed below with aryl groups.

Aryl: The term “aryl,” as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 3 to 20 ring atoms (unlessotherwise specified). Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g., C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆aryl,” as used herein,pertains to an aryl group having 5 or 6 ring atoms. Examples of groupsof aryl groups include C₃₋₂₀aryl, C₃₋₁₂aryl, C₅₋₁₂aryl, C₅₋₇aryl, aC₅₋₆aryl.

The ring atoms may be all carbon atoms, as in “carboaryl groups” (e.g.,C₅₋₂₀carboaryl).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups” (e.g., C₅₋₂₀heteroaryl).

Heterocyclic groups (including heteroaryl groups) which have a nitrogenring atom in the form of an —NH— group may be N-substituted, that is, as—NR—. For example, pyrrole may be N-methyl substituted, to giveN-methypyrrole. Examples of N-substituents include, but are not limitedto C₁₋₇alkyl, C₃₋₂₀heterocyclyl, C₅₋₂₀aryl, and acyl groups.

Heterocyclic groups (including heteroaryl groups) which have a nitrogenring atom in the form of an —N═ group may be substituted in the form ofan N-oxide, that is, as —N(→O)═ (also denoted —N⁺(→O⁻)═). For example,quinoline may be substituted to give quinoline N-oxide; pyridine to givepyridine N-oxide; benzofurazan to give benzofurazan N-oxide (also knownas benzofuroxan).

Cyclic groups may additionally bear one or more oxo (═O) groups on ringcarbon atoms.

The above alkyl, heterocyclyl, and aryl groups, whether alone or part ofanother substituent, may themselves optionally be substituted with oneor more groups selected from themselves and the additional substituentslisted below.

Hydrogen: —H. Note that if the substituent at a particular position ishydrogen, it may be convenient to refer to the compound as being“unsubstituted” at that position.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇alkylgroup (also referred to as a C₁₋₇alkoxy group, discussed below), aC₃₋₂₀heterocyclyl group (also referred to as a C₃₋₂₀heterocyclyloxygroup), or a C₅₋₂₀aryl group (also referred to as a C₅₋₂₀aryloxy group),preferably a C₁₋₇alkyl group.

C₁₋₇alkoxy: —OR, wherein R is a C₁₋₇alkyl group. Examples of C₁₋₇alkoxygroups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy),—O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu)(sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).

Oxo (keto, -one): ═O.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇alkyl group (also referred to as C₁₋₇alkylacyl or C₁₋₇alkanoyl), aC₃₋₂₀heterocyclyl group (also referred to as C₃₋₂₀heterocyclylacyl), ora C₅₋₂₀aryl group (also referred to as C₅₋₂₀arylacyl), preferably aC₁₋₇alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇alkyl group, aC₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of acyloxygroups include, but are not limited to, —OC(═O)CH₃ (acetoxy),—OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarbonyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀arylgroup, preferably a C₁₋₇alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, ora C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group, and R² isan acyl substituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclylgroup, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group.Examples of acylamide groups include, but are not limited to,—NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may together forma cyclic structure, as in, for example, succinimidyl, maleimidyl, andphthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇alkyl group (also referred to asC₁₋₇alkylamino or di-C₁₋₇alkylamino), a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group, or, in the case of a“cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Nitro: —NO₂.

Cyano (nitrile, carbonitrile): —CN.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkylgroup. Examples of C₁₋₇alkylthio groups include, but are not limited to,—SCH₃ and —SCH₂CH₃.

Silyl: —SiR₃, where R is a silyl substituent, for example, —H, aC₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group,preferably —H, a C₁₋₇alkyl group, or a C₅₋₂₀aryl group. Examples ofsilyl groups include, but are not limited to, —SiH₃, —SiH₂(CH₃),—SiH(CH₃)₂, —Si(CH₃)₃, —Si(Et)₃, —Si(iPr)₃, —Si(tBu)(CH₃)₂, and—Si(tBu)₃.

In many cases, substituents may themselves be substituted. For example,a C₁₋₇alkyl group may be substituted with, for example, hydroxy (alsoreferred to as a C₁₋₇hydroxyalkyl group), C₁₋₇alkoxy (also referred toas a C₁₋₇alkoxyalkyl group), amino (also referred to as a C₁₋₇aminoalkylgroup), halo (also referred to as a C₁₋₇haloalkyl group), carboxy (alsoreferred to as a C₁₋₇carboxyalkyl group), and C₅₋₂₀aryl (also referredto as a C₅₋₂₀aryl-C₁₋₇alkyl group).

Similarly, a C₅₋₂₀aryl group may be substituted with, for example,hydroxy (also referred to as a C₅₋₂₀hydroxyaryl group), halo (alsoreferred to as a C₅₋₂₀haloaryl group), amino (also referred to as aC₅₋₂₀aminoaryl group, e.g., as in aniline), C₁₋₇alkyl (also referred toas a C₁₋₇alkyl-C₅₋₂₀aryl group, e.g., as in toluene), and C₁₋₇alkoxy(also referred to as a C₁₋₇alkoxy-C₅₋₂₀aryl group, e.g., as in anisole).

These and other specific examples of such substituted-substituents aredescribed below.

C₁₋₇haloalkyl group: The term “C₁₋₇haloalkyl group,” as used herein,pertains to a C₁₋₇alkyl group in which at least one hydrogen atom (e.g.,1, 2, 3) has been replaced with a halogen atom (e.g., F, Cl, Br, I). Ifmore than one hydrogen atom has been replaced with a halogen atom, thehalogen atoms may independently be the same or different. Every hydrogenatom may be replaced with a halogen atom, in which case the group mayconveniently be referred to as a C₁₋₇perhaloalkyl group.” Examples ofC₁₋₇haloalkyl groups include, but are not limited to, —CF₃, —CHF₂,—CH₂F, —CCl₃, —CBr₃, —CH₂CH₂F, —CH₂CHF₂, and —CH₂CF₃.

C₁₋₇haloalkoxy: —OR, wherein R is a C₁₋₇haloalkyl group. Examples ofC₁₋₇haloalkoxy groups include, but are not limited to, —OCF₃, —OCHF₂,—OCH₂F, —OCCl₃, —OCBr₃, —OCH₂CH₂F, —OCH₂CHF₂, and —OCH₂CF₃.

C₁₋₇hydroxyalkyl: The term “C₁₋₇hydroxyalkyl group,” as used herein,pertains to a C₁₋₇alkyl group in which at least one hydrogen atom hasbeen replaced with a hydroxy group. Examples of C₁₋₇hydroxyalkyl groupsinclude, but are not limited to, —CH₂OH, —CH₂CH₂OH, and —CH(OH)CH₂OH.

C₁₋₇carboxyalkyl: The term “C₁₋₇carboxyalkyl group,” as used herein,pertains to a C₁₋₇alkyl group in which at least one hydrogen atom hasbeen replaced with a carboxy group. Examples of C₁₋₇carboxyalkyl groupsinclude, but are not limited to, —CH₂COOH and —CH₂CH₂COOH.

C₁₋₇aminoalkyl: The term “C₁₋₇aminoalkyl group,” as used herein,pertains to a C₁₋₇alkyl group in which at least one hydrogen atom hasbeen replaced with an amino group. Examples of C₁₋₇aminoalkyl groupsinclude, but are not limited to, —CH₂NH₂, —CH₂CH₂NH₂, and—CH₂CH₂N(CH₃)₂.

C₁₋₇aminoalkylamino: The term “C₁₋₇aminoalkylamino,” as used herein,pertains to an amino group, —NR¹R², in which one of the substituents, R¹or R², is itself a C₁₋₇aminoalkyl group (—C₁₋₇alkyl-NR¹R²). TheC₁₋₇aminoalkylamino may be represented, for example, by the formula—NR¹—C₁₋₇alkyl-NR¹R². Examples of amino-C₁₋₇alkylamino groups include,but are not limited to, groups of the formula —NR¹(CH₂)_(n)NR¹R², wheren is 1 to 6, for example, —NHCH₂NH₂, —NH(CH₂)₂NH₂, —NH(CH₂)₃NH₂,—NH(CH₂)₄NH₂, —NH(CH₂)₅NH₂, —NH(CH₂)₆NH₂, —NHCH₂NH(Me), —NH(CH₂)₂NH(Me),—NH(CH₂)₃NH(Me), —NH(CH₂)₄NH(Me), —NH(CH₂)₅NH(Me), —NH(CH₂)₆NH(Me),—NHCH₂NH(Et), —NH(CH₂)₂NH(Et), —NH(CH₂)₃NH(Et), —NH(CH₂)₄NH(Et),—NH(CH₂)₅NH(Et), and —NH(CH₂)₆NH(Et).

C₁₋₇alkyl-C₅₋₂₀aryl: The term “C₁₋₇alkyl-C₅₋₂₀aryl,” as used herein,describes certain C₅₋₂₀aryl groups which have been substituted with aC₁₋₇alkyl group. Examples of such groups include, but are not limitedto, tolyl (from toluene), xylyl (from xylene), mesityl (frommesitylene), and cumenyl (or cumyl, from cumene), and duryl (fromdurene).

C₁₋₇alkyl-C₅₋₂₀aryloxy: The term “C₁₋₇alkyl-C₅₋₂₀aryloxy,” as usedherein, describes certain C₅₋₂₀aryloxy groups which have beensubstituted with a C₁₋₇alkyl group. Examples of such groups include, butare not limited to, tolyloxy, xylyloxy, mesityloxy, cumenyloxy, andduryloxy.

C₅₋₂₀aryl-C₁₋₇alkyl: The term “C₅₋₂₀aryl-C₁₋₇alkyl,” as used herein,describers certain C₁₋₇alkyl groups which have been substituted with aC₅₋₂₀aryl group. Examples of such groups include, but are not limitedto, benzyl (phenylmethyl, PhCH₂—), benzhydryl (Ph₂CH—), trityl(triphenylmethyl, Ph₃C—), phenethyl (phenylethyl, Ph-CH₂CH₂—), styryl(Ph-CH═CH—), cinnamyl (Ph-CH═CH—CH₂—).

C₅₋₂₀aryl-C₁₋₇alkoxy: The term “C₅₋₂₀aryl-C₁₋₇alkoxy,” as used herein,describes certain C₁₋₇alkoxy groups which have been substituted with aC₅₋₂₀aryl group. Examples of such groups include, but are not limitedto, benzyloxy, benzhydryloxy, trityloxy, phenethoxy, styryloxy, andcimmamyloxy.

C₅₋₂₀haloaryl: The term “C₅₋₂₀haloaryl,” as used herein, describescertain C₅₋₂₀aryl groups which have been substituted with one or morehalo groups. Examples of such groups include,-but are not limited to,halophenyl (e.g., fluorophenyl, chlorophenyl, bromophenyl, oriodophenyl, whether ortho-, meta-, or para-substituted), dihalophenyl,trihalophenyl, tetrahalophenyl, and pentahalophenyl.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Isomers, Salts, Solvates, Protected Forms, and Prodrugs

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers,” as used herein, are structural (orconstitutional) isomers (i.e., isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms failing within that class (e.g., C₁₋₇alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.,asymmetric synthesis) and separation (e.g., fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound alsoincludes ionic, salt, solvate, and protected forms of thereof, forexample, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1–19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.,active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form. The term “chemicallyprotected form” is used herein in the conventional chemical sense andpertains to a compound in which one or more reactive functional groupsare protected from undesirable chemical reactions under specifiedconditions (e.g., pH, temperature, radiation, solvent, and the like). Inpractice, well known chemical methods are employed to reversibly renderunreactive a functional group, which otherwise would be reactive, underspecified conditions. In a chemically protected form, one or morereactive functional groups are in the form of a protected or protectinggroup (also known as a masked or masking group or a blocked or blockinggroup). By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley andSons, 1999).

A wide variety of such “protecting,” “blocking,” or “masking” methodsare widely used and well known in organic synthesis. For example, acompound which has two nonequivalent reactive functional groups, both ofwhich would be reactive under specified conditions, may be derivatizedto render one of the functional groups “protected,” and thereforeunreactive, under the specified conditions; so protected, the compoundmay be used as a reactant which has effectively only one reactivefunctional group. After the desired reaction (involving the otherfunctional group) is complete, the protected group may be “deprotected”to return it to its original functionality.

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl ort-butyldimethylsilyl ether, or an acetyl ester (—OC(═O)CH₃, —OAc).

For example, an aldehyde or ketone group may be protected as an acetal(R—CH(OR)₂) or ketal (R₂C(OR)₂), respectively, in which the carbonylgroup (>C═O) is converted to a diether (>C(OR)₂), by reaction with, forexample, a primary alcohol. The aldehyde or ketone group is readilyregenerated by hydrolysis using a large excess of water in the presenceof acid.

For example, an amine group may be protected, for example, as an amide(—NRCO—R) or a urethane (—NRCO—OR), for example, as: a methyl amide(—NHCO—CH₃); a benzyloxy amide (—NHCO—OCH₂C₆H₅, —NH—Cbz); as a t-butoxyamide (—NHCO—OC(CH₃)₃, —NH—Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH—Bpoc), as a 9-fluorenylmethoxy amide(—NH—Fmoc), as a 6-nitroveratryloxy amide (—NH—Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a2(-phenylsulphonyl)ethyloxy amide (—NH—Psec); or, in suitable cases(e.g., cyclic amines), as a nitroxide radical (>N—O.).

For example, a carboxylic acid group may be protected as an ester forexample, as: an C₁₋₇alkyl ester (e.g., a methyl ester; a t-butyl ester);a C₁₋₇haloalkyl ester (e.g., a C₁₋₇trihaloalkyl ester); atriC₁₋₇alkylsilyl-C₁₋₇alkyl ester; or a C₅₋₂₀aryl-C₁₋₇alkyl ester (e.g.benzyl ester; a nitrobenzyl ester); or as an amide, for example, as amethyl amide.

For example, a thiol group may be protected as a thioether (—SR), forexample, as: a benzyl thioether; an acetamidomethyl ether(—S—CH₂NHC(═O)CH₃).

Acronyms

For convenience, many chemical moieties are represented using well knownabbreviations, including but not limited to, methyl (Me), ethyl (Et),n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu), sec-butyl (sBu),iso-butyl (iBu), tert-butyl (tBu), n-hexyl (nHex), cyclohexyl (cHex),phenyl (Ph), biphenyl (biPh), benzyl (Bn), naphthyl (naph), methoxy(MeO), ethoxy (EtO), benzoyl (Bz), and acetyl (Ac).

Synthesis

Several methods for the chemical synthesis of compounds of the presentinvention are described herein. These and/or other well known methodsmay be modified and/or adapted in known ways in order to facilitate thesynthesis of additional compounds within the scope of the presentinvention.

In one approach, 2- or 4-fluoronitrobenzene (1) is reacted withdiethanolamine (2) to form the corresponding 2- or4-(di(2-hydroxyethyl)amino)nitrobenzene (3).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) heating at 130° C., without solvent, 16 h,83%, purification by column chromatography (Kiselgel 60, eluent: AcOEt).

The hydroxy groups of the product (3) are protected ast-butyldimethylsilyl ethers (4). The nitro group is then reduced to givean amino group (5), which is first mono-protected, for example, as abenzyl carbamate (6), then substituted (e.g., alkylated) (7), and thendeprotected to give the corresponding N-substituted product (8).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) t-butyl-dimethyl-silyl chloride(TBDMSiCl), imidazole, dimethyl formamide (DMF), room temperature(r.t.), 20 h, 89%; (ii) H₂, Pd/C (10%), THF, r.t., 6 h, 95%; (iii)N-benzyloxycarbonyloxy-succinimide, THF, r.t., 16 h, 95%; (iv) alkylhalide (e.g., alkyl iodide, e.g., methyl iodide), NaH, THF, r.t., 12 h,100%; and (v) H₂, Pd/C 10%, AcOEt, r.t., 3 h, 100%.

Another aspect of the present invention pertains to the intermediates 7and 8 (and silyl analogs, —SiR₃, of —SiTBDM) in the above scheme, whichare suitable for use in the methods of synthesis described herein.Another aspect of the present invention pertains to the use of suchintermediates, as described herein, in the methods of synthesisdescribed herein.

Separately, a linker compound (18), which incorporates the CPG2substrate and the self-immolative core, is prepared, reacted with4-nitrophenyl chloroformate to produce an activated linker (19), whichwas subsequently reacted with the N-substituted compound (8) (seebelow).

The linker compound (18) may be prepared, for example by reactionbetween an amine and an isocyanate.

In a first approach, (a), the self-immolative core reagent bears anamine (and has an unprotected hydroxy group) (9) and the glutamatereagent bears an isocyanate (16). The resulting product is the diesterof the linker compound (18), which is then activated by formation of thecorresponding glutamate-urea-benzyl-p-nitrophenylcarbonate (19).

In a second approach, (b), the self-immolative core reagent bears anamine (and has a protected hydroxy group) (12) and the glutamate reagentbears an isocyanate (16). The resulting product is the hydroxy-protecteddiester of the linker compound (17), which is then hydroxy-deprotectedto give the diester of the linker compound (18), which is then convertedto the reactive linker compound (19).

In a third approach, (c) the self-immolative core reagent bears anisocyanate (and has a protected hydroxy group) (13) and the glutamatereagent bears an amine (15). The resulting product is thehydroxy-protected diester of the linker compound (17), which is thenhydroxy-deprotected to give the diester of the linker compound (18),which is then converted to the reactive linker compound (19).

4-aminobenzyl alcohols (9) (suitable for use in approach (a)) areavailable commercially (for example, from Lancaster; Fluka; etc.) or maybe synthesised using well known methods.

Hydroxy-protected 4-aminobenzyl alcohols (12) (suitable for approach(b)) and hydroxy-protected 4-isocyanatobenzyl alcohols (13) (suitablefor appro ach (c)) may be prepared as follows. The hydroxy group ofoptionally substituted p-nitrobenzyl alcohol (10) is first protected,for example, as a t-butyldiphenylsilyl ether or a 2-tetrahydropyranylether (11). The nitro group is then reduced to form an amino group (12),and the amino group is then converted to an isocyanato group (13).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) tert-butyldiphenylsilylchloride (TBDPSiCl)(then R¹ is —Si(tBu)(Ph)₂), imidazole, DMF (or THF); or 3,4-dihydropyran(then R¹ is 2-tetrahydropyranyl), pyridinium-p-toluene sulphonic acid(PPTS), CH₂Cl₂, room temperature; (ii) H₂, Pd/C (10%), HCO₂NH₄, EtOH;(iii) (Cl₃CO)₂CO, NEt₃, toluene, 70° C.

Separately, the acid groups of glumatic acid (14) are protected, forexample, as ester groups (15), for example, as the di-allyl ester or thedi-t-butyl ester, preferably as the di-allyl ester.

An example of such a method is illustrated in the following scheme, inwhich the conditions are suitable esterification conditions. Forexample, where R^(G) is tBu, suitable conditions are: (i) isobutene,H₂SO₄, DCM, −70° C. to r.t., 16 h, 86% (the yield is better than the onereported in the literature; see, for example, Ferenz et al., 1989). Notealso that the di-tBu ester is commercially available, but expensive(bis-t-Bu-L-glutamate, HCl salt, 25 g, £257, NovaBiochem UK, CNBiosciences (UK) Ltd.), and that the preferred di-allyl ester iscommercially available and cheap (bis-allyl-L-glutamate, tosylate salt,25 g, £64, NovaBiochem).

If necessary, the amino group of the protected glutamic acid (15) isconverted to an isocyanato group (16) (suitable for approaches (a) and(b)).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) (Cl₃CO)₂CO, NEt₃, toluene or other aproticsolvent such as THF or dichloromethane (DCM), −78° C.

As described above, in approach (a), a self-immolative core reagentbearing an amine (and having an unprotected hydroxy group) (9) isreacted with a glutamate reagent bearing an isocyanate (16), to give theglutamate-urea-benzyl-ether (18).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) THF, NEt₃, room temperature.

As described above, in approach (b), a self-immolative core reagentbearing an amine (and having a protected hydroxy group) (12) is reactedwith a glutamate reagent bearing an isocyanate (16), to give thehydroxy-protected glutamate-urea-benzyl-ether (17).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) THF, NEt₃, room temperature.

As described above, in approach (c), a self-immolative core reagentbearing an isocyanate (and having a protected hydroxy group) (13) isreacted with a glutamate reagent bearing an amine (15), to give thehydroxy-protected glutamate-urea-benzyl-ether (17).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) THF, NEt₃, room temperature.

The hydroxy-protecting group of the hydroxy-protectedglutamate-urea-benzyl-ether (17) is then removed using suitabledeprotection conditions to yield the correspondingglutamate-urea-benzyl-alcohol (18).

An example of such a method is illustrated in the following scheme, inwhich suitable deprotection conditions may be used. For example, when R¹is TBDPSi, the conditions are: (i) Bu₄NF, THF, room temperature; when R¹is 2-tetrahydropyranyl (THP), the conditions are: (i) AcOH, THF, H₂O.Note that, when R¹ is TBDPSi, R^(E) should not be allyl (but R^(E) maybe, e.g., t-butyl), because allyl ester-deprotection leads to undesiredby-products; when R¹ is THP, such problems do not arise (and R^(E) maybe, e.g., allyl, t-butyl, etc.).

The glutamate-urea-benzyl-alcohol (18) is then activated by formation ofthe corresponding glutamate-urea-benzyl-p-nitrophenylcarbonate (19).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) 4-nitrophenylchloroformate, THF (orCH₃CN), NEt₃, room temperature, 1 h, 48–50%.

As described above, the reactive linker compound (19) is then reactedwith the N-substituted compound (8), to form the conjugate (20).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) dimethyl acetamide (DMA), r.t., 5 days,28%.

The t-butyldimethylsilyl ether groups of the conjugate (20) are thenremoved to give the corresponding compound (21) bearing hydroxy groups,which are then converted to sulfonates (e.g., mesylates) (22), and thento a halides (23).

An example of such a method is illustrated in the following scheme, inwhich the conditions are: (i) NEt₃.3HF, THF, r.t., 7 h, 97%; (ii) Mes₂O,NEt₃, 4-N,N-dimethylaminopyridine (DMAP), CH₂Cl₂, r.t., 2.5 h, 99%; and(iii) where X is I: Nal, acetone, reflux, 4 h, 94%; where X is Br: LiBr,THF, reflux, 1.5 h, 69%; and where X is Cl: LiCl, dimethyl acetamide(DMA), r.t., 24 h, 51%.

Another aspect of the present invention pertains to the intermediates20, 21, 22, and 23 in the above scheme (and silyl, —SiR₃₁ analogs of—SiTBDM; and sulfonyl, —SO₂R, analogs of —SO₂Me), which are suitable foruse in the methods of synthesis described herein.

Another aspect of the present invention pertains to the use of suchintermediates, as described herein, in the methods of synthesisdescribed herein.

The glutamic acid ester groups of the halide (23) are then removed usingsuitable deprotection conditions, to give the glutamic acid product(24).

An example of such a method is illustrated in the following scheme, inwhich suitable deprotection conditions are used. For example, whereR^(G) is allyl, the conditions are: (i) Pd(PPh₃)₄, morpholine orpyrrolidine, CH₂Cl₂, 50 min, then passed through an ion-exchange-column(e.g., Amberlite IRC50, weakly acidic), eluent MeOH, 84%.

Uses

The present invention provides compounds (prodrugs) as described herein.

One aspect of the present invention pertains to compounds (prodrugs)which (a) regulate (e.g., inhibit) cell proliferation; (b) inhibit cellcycle progression; (c) promote apoptosis; or (d) a combination of one ormore of these.

One aspect of the present invention pertains to a method of (a)regulating (e.g., inhibiting) proliferation of a cell; (b) inhibitingcell cycle progression of a cell; (c) promoting apoptosis of a cell; or(d) a combination of one or more of these, in vitro or in vivo,comprising contacting the cell with an effective amount of a compound(prodrug), as described herein.

One aspect of the present invention pertains to a method of regulating(e.g., inhibiting) proliferation of a cell, in vitro or in vivo,comprising contacting the cell with an effective amount of a compound(prodrug), as described herein.

In one embodiment, the method is performed in vitro.

In one embodiment, the method is performed in vivo.

Preferably, the compound (prodrug) is provided in the form of apharmaceutically acceptable composition.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g., bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound regulates (e.g., inhibits) cell proliferation,etc. For example, assays which may conveniently be used to assess theactivity offered by a particular compound (prodrug) are described in theexamples below.

For example, a sample of cells (e.g., from a tumour) may be grown invitro and a compound (prodrug) brought into contact with said cells, andthe effect of the compound on those cells observed. As an example of“effect,” the morphological status of the cells (e.g., alive or dead,etc.) may be determined. Where the active compound is found to exert aninfluence on the cells, this may be used as a prognostic or diagnosticmarker of the efficacy of the compound in methods of treating a patientcarrying cells of the same cellular type.

Methods of Treatment, Etc.

One aspect of the present invention pertains to a method of treatment,for example, of cancer, a proliferative condition, or other condition asdescribed herein, comprising administering to a subject in need oftreatment a therapeutically-effective amount of a compound (prodrug), asdescribed herein, preferably in the form of a pharmaceuticalcomposition.

One aspect of the present invention pertains to a compound (prodrug) foruse in a method of treatment of the human or animal body by therapy, forexample, in the treatment of cancer, a proliferative condition, or othercondition as described herein.

One aspect of the present invention pertains to the use of a compound(prodrug) for the manufacture of a medicament, for example, for thetreatment of cancer, a proliferative condition, or other condition asdescribed herein.

One aspect of the present invention pertains to a method formanufacturing a medicament intended for therapeutic application, forexample, for the treatment of cancer, a proliferative condition, orother condition as described herein, characterised in that a compound(prodrug), as described herein, is used.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,and cure of the condition. Treatment as a prophylactic measure (i.e.,prophylaxis) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound (prodrug), or a material, composition ordosage form comprising a compound (prodrug), which is effective forproducing some desired therapeutic effect, commensurate with areasonable benefit/risk ratio.

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g., drugs, antibodies (e.g., as inimmunotherapy), prodrugs (e.g., as in photodynamic therapy, enzymeprodrug therapy (EPT), such as GDEPT, ADEPT, etc.)); surgery; radiationtherapy; and gene therapy.

Compound (prodrugs) may also be used, as described above, in combinationtherapies, that is, in conjunction with other agents, for example,cytotoxic agents.

Antiproliferative Applications

The present invention also provides compounds (prodrugs) which areantiproliferative agents.

The term “antiproliferative agent” as used herein, pertain to a compoundwhich treats a proliferative condition (i.e., a compound which is usefulin the treatment of a proliferative condition).

The terms “proliferative condition,” “proliferative disorder,” and“proliferative disease,” are used interchangeably herein and pertain toan unwanted or uncontrolled cellular proliferation of excessive orabnormal cells which is undesired, such as, neoplastic or hyperplasticgrowth.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g., lung cancer, small cell lungcancer, gastrointestinal cancer, bowel cancer, colon cancer, breastcarcinoma, ovarian carcinoma, prostate cancer, testicular cancer, livercancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer,sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias,psoriasis, bone diseases, fibroproliferative disorders (e.g., ofconnective tissues), and atherosclerosis.

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

Anticancer Applications

The present invention also provides compounds (prodrugs) which areanticancer agents.

The term “anticancer agent” as used herein, pertains to a compound(prodrug) which treats a cancer (i.e., a compound which is useful in thetreatment of a cancer). The anti-cancer effect may arise through one ormore mechanisms, including but not limited to, the regulation of cellproliferation, the inhibition of cell cycle progression, the inhibitionof angiogenesis (the formation of new blood vessels), the inhibition ofmetastasis (the spread of a tumour from its origin), the inhibition ofinvasion (the spread of tumour cells into neighbouring normalstructures), or the promotion of apoptosis (programmed cell death).

Examples of cancers are discussed herein.

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound treats a cancerous condition for any particularcell type. For example, assays which may conveniently be used to assessthe activity offered by a particular compound are described in theexamples below.

Enzyme Prodrug Therapy Etc.

As discussed above, the compounds (prodrugs) described herein are usefulin enzyme prodrug therapy (EPT) and related methods.

One aspect of the present invention pertains to a method of enzymeprodrug therapy (EPT) which employs a compound (prodrug), as describedherein, and a carboxypeptidase enzyme, as described herein.

One aspect of the present invention pertains to a method of (a)regulating (e.g., inhibiting) proliferation of a cell; (b) inhibitingcell cycle progression of a cell; (c) promoting apoptosis of a cell; or(d) a combination of one or more of these, in vitro or in vivo,comprising contacting the cell with an effective amount of a compound(prodrug), as described herein, in the presence of a carboxypeptidaseenzyme, as described herein.

One aspect of the present invention pertains to a method of regulating(e.g., inhibiting) proliferation of a cell, in vitro or in vivo,comprising contacting the cell with an effective amount of a compound(prodrug), as described herein, in the presence of a carboxypeptidaseenzyme, as described herein.

One aspect of the present invention pertains to a method of treatment,for example, of cancer, a proliferative condition, or other condition asdescribed herein, comprising administering to a subject in need oftreatment a therapeutically-effective amount of a compound (prodrug), asdescribed herein, preferably in the form of a pharmaceuticalcomposition, in the presence of a carboxypeptidase enzyme, as describedherein.

In one embodiment, the enzyme is a bacterial carboxypeptidase enzyme.

In one embodiment, the enzyme is CPG2.

In one embodiment, the enzyme is bacterial CPG2.

Examples of suitable carboxypeptidase enzymes, and methods for theirpreparation and use, are well known in the art. Guidance for theselection of additional suitable carboxypeptidase enzymes, and methodsfor their preparation and use, is also widely available. See, forexample, the discussion above under the heading “Background,” and thedocuments cited therein.

Antibody Directed Enzyme Prodrug Therapy Etc.

As discussed above, the compounds (prodrugs) described herein may beused in a method of antibody directed enzyme prodrug therapy (ADEPT) andrelated methods.

One aspect of the present invention pertains to a method of antibodydirected enzyme prodrug therapy (ADEPT) which employs a compound(prodrug), as described herein, and a carboxypeptidase enzyme, asdescribed herein.

One aspect of the present invention pertains to a two component system(comprising two components for use in association with one another),comprising: (a) a prodrug, as described herein; and (b) an antibody orfragment thereof conjugated or fused to a carboxypeptidase enzyme, asdescribed herein.

One aspect of the present invention pertains to a two component system,as described above, for use in a method of treatment of the human oranimal body by therapy.

One aspect of the present invention pertains to use of a two componentsystem, as described above, for the manufacture of a medicament for thetreatment of, for example, cancer, a proliferative condition, or othercondition as described herein.

One aspect of the invention pertains to a kit comprising (a) a prodrug,as described herein; (b) an antibody or fragment thereof conjugated orfused to a carboxypeptidase enzyme, as described herein; and (c)instructions for use, for example, written instructions on how toperform ADEPT.

One aspect of the present invention pertains to a method of (a)regulating (e.g., inhibiting) proliferation of a cell; (b) inhibitingcell cycle progression of a cell; (c) promoting apoptosis of a cell; or(d) a combination of one or more of these, in vitro or in vivo,comprising: (i) contacting the cell with an antibody or fragment thereofconjugated or fused to a carboxypeptidase enzyme, as described herein;and (ii) contacting the cell with a therapeutically-effective amount ofa compound (prodrug), as described herein, preferably in the form of apharmaceutical composition.

One aspect of the present invention pertains to a method of regulating(e.g., inhibiting) proliferation of a cell, in vitro or in vivo,comprising: (i) contacting the cell with an antibody or fragment thereofconjugated or fused to a carboxypeptidase enzyme, as described herein;and (ii) contacting the cell with a therapeutically-effective amount ofa compound (prodrug), as described herein, preferably in the form of apharmaceutical composition.

One aspect of the present invention pertains to a method of treatment,for example, of cancer, a proliferative condition, or other condition asdescribed herein, comprising administering to a subject in need oftreatment: (i) an antibody or fragment thereof conjugated or fused to acarboxypeptidase enzyme, as described herein; and (ii) atherapeutically-effective amount of a compound (prodrug), as describedherein, preferably in the form of a pharmaceutical composition.

Examples of suitable antibodies and antibody conjugates, and methods fortheir preparation and use, including methods of ADEPT, are well known inthe art. Guidance for the selection of additional suitable antibodiesand antibody conjugates, and methods for their preparation and use, isalso widely available. See, for example, the discussion above under theheading “Background,” and the documents cited therein.

Gene Directed Enzyme Prodrug Therapy Etc.

As discussed above, the compounds (prodrugs) described herein may beused in a method of gene directed enzyme prodrug therapy (GDEPT) andrelated methods.

One aspect of the present invention pertains to a method of genedirected enzyme prodrug therapy (GDEPT) which employs a compound(prodrug), as described herein, and a carboxypeptidase enzyme, asdescribed herein.

One aspect of the present invention pertains to a two component system(comprising two components for use in association with one another),comprising: (a) a prodrug, as described herein; and (b) a nucleic acidencoding (e.g., as part of a vector capable of expressing) acarboxypeptidase enzyme,.as described herein.

One aspect of the present invention pertains to a two component system,as described above, for use in a method of treatment of the human oranimal body by therapy.

One aspect of the present invention pertains to use of a two componentsystem, as described above, for the manufacture of a medicament for thetreatment of, for example, cancer, a proliferative condition, or othercondition as described herein.

One aspect of the invention pertains to a kit comprising (a) a prodrug,as described herein; (b) a nucleic acid encoding (e.g., as part of avector capable of expressing) a carboxypeptidase enzyme, as describedherein; and (c) instructions for use, for example, written instructionson how to perform GDEPT.

One aspect of the present invention pertains to a method of (a)regulating (e.g., inhibiting) proliferation of a cell; (b) inhibitingcell cycle progression of a cell; (c) promoting apoptosis of a cell; or(d) a combination of one or more of these, in vitro or in vivo,comprising: (i) contacting the cell with a nucleic acid encoding (e.g.,as part of a vector capable of expressing) a carboxypeptidase enzyme, asdescribed herein; and (ii) contacting the cell with atherapeutically-effective amount of a compound (prodrug), as describedherein, preferably in the form of a pharmaceutical composition.

One aspect of the present invention pertains to a method of regulating(e.g., inhibiting) proliferation of a cell, in vitro or in vivo,comprising: (i) contacting the cell with a nucleic acid encoding (e.g.,as part of a vector capable of expressing) a carboxypeptidase enzyme, asdescribed herein; and (ii) contacting the cell with atherapeutically-effective amount of a compound (prodrug), as describedherein, preferably in the form of a pharmaceutical composition.

One aspect of the present invention pertains to a method of treatment,for example, of cancer, a proliferative condition, or other condition asdescribed herein, comprising administering to a subject in need oftreatment: (i) a nucleic acid encoding (e.g., as part of a vectorcapable of expressing) a carboxypeptidase enzyme, as described herein;and (ii) a therapeutically-effective amount of a compound (prodrug), asdescribed herein, preferably in the form of a pharmaceuticalcomposition.

Examples of suitable nucleic acids, vectors, and methods for theirpreparation and use, including methods of GDEPT, are well known in theart. Guidance for the selection of additional suitable vectors, andmethods for their preparation and use, is also widely available. See,for example, the discussion above under the heading “Background,” andthe documents cited therein.

Additional Uses

The compounds (prodrugs) may also be used as cell culture additives, forexample, in order to regulate (e.g., inhibit) cell proliferation invitro.

The compounds (prodrugs) may also be used as part of an in vitro assay,for example, in order to determine whether a candidate host is likely tobenefit from treatment with the compound in question.

The compounds (prodrugs) may also be used as a standard, for example, inan assay, in order to identify other compounds (prodrugs), other drugs,other anticancer agents, other antiproliferative agents, etc.

Routes of Administration

The compound (prodrug) or pharmaceutical composition comprising thecompound (prodrug) may be administered to a subject by any convenientroute of administration, whether systemically/ peripherally or topically(i.e., at the site of desired action).

Routes of administration include, but are not limited to, oral (e.g, byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal; by implant of a depot or reservoir, for example,subcutaneously or intramuscularly.

The Subject

In one embodiment, the subject is a prokaryote (e.g., bacteria) or aeukaryote (e.g., protoctista, fungi, plants, animals).

In one embodiment, the subject is an animal.

In one embodiment, the subject is a chordate, a vertebrate, a mammal, abird, a reptile (e.g., snakes, lizards, crocodiles), an amphibian (e.g.,frogs, toads), a bony fish (e.g., salmon, plaice, eel, lungfish), acartilaginous fish (e.g., sharks, rays), or a jawless fish (e.g.,lampreys, hagfish).

In one embodiment, the subject is a mammal.

In one embodiment, the subject is a mammal, a placental mammal, amarsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

In one embodiment, the subject is a human.

The subject may be any of its forms of development, for example, aspore, a seed, an egg, a larva, a pupa, or a foetus.

Formulations

While it is possible for the compound (prodrug) to be used (e.g.,administered) alone, it is often preferable to present it as aformulation.

Thus, one aspect of the present invention pertains to a compositioncomprising a compound (prodrug), as described herein, and a carrier.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a compound(prodrug), as described herein, and a pharmaceutically acceptablecarrier.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one compound (prodrug), as described herein,together with one or more other pharmaceutically acceptable ingredientswell known to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton,Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one compound(prodrug), as defined above, together with one or more otherpharmaceutically acceptable ingredients well known to those skilled inthe art, e.g., carriers, diluents, excipients, etc. If formulated asdiscrete units (e.g., tablets, etc.), each unit contains a predeterminedamount (dosage) of the compound (prodrug).

The term “pharmaceutically acceptable” as used herein pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound (prodrug) with a carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the compound(prodrug) with carriers (e.g., liquid carriers, finely divided solidcarrier, etc.), and then shaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations may suitably be in the form of liquids, solutions (e.g.,aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups,electuaries, mouthwashes, drops, tablets (including, e.g., coatedtablets), granules, powders, lozenges, pastilles, capsules (including,e.g., hard and soft gelatin capsules), cachets, pills, ampoules,boluses, suppositories, pessaries, tinctures, gels, pastes, ointments,creams, lotions, oils, foams, sprays, mists, or aerosols.

Formulations may suitably be provided as a patch, adhesive plaster,bandage, dressing, or the like which is impregnated with one or moreactive compounds and optionally one or more other pharmaceuticallyacceptable ingredients, including, for example, penetration, permeation,and absorption enhancers. Formulations may also suitably be provided ina the form of a depot or reservoir.

The compound (prodrug) may be dissolved in, suspended in, or admixedwith one or more other pharmaceutically acceptable ingredients. Thecompound (prodrug) may be presented in a liposome or othermicroparticulate which is designed to target the compound (prodrug), forexample, to blood components or one or more organs.

Formulations suitable for oral administration (e.g, by ingestion)include liquids, solutions (e.g., aqueous, non-aqueous), suspensions(e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water,water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders,capsules, cachets, pills, ampoules, boluses.

Formulations suitable for buccal administration include mouthwashes,lozenges, pastilles, as well as patches, adhesive plasters, depots, andreservoirs. Lozenges typically comprise the compound (prodrug) in aflavored basis, usually sucrose and acacia or tragacanth. Pastillestypically comprise the compound (prodrug) in an inert matrix, such asgelatin and glycerin, or sucrose and acacia. Mouthwashes typicallycomprise the compound (prodrug) in a suitable liquid carrier.

Formulations suitable for sublingual administration include tablets,lozenges, pastilles, capsules, and pills.

Formulations suitable for oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),mouthwashes, lozenges, pastilles, as well as patches, adhesive plasters,depots, and reservoirs.

Formulations suitable for non-oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),suppositories, pessaries, gels, pastes, ointments, creams, lotions,oils, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for transdermal administration include gels,pastes, ointments, creams, lotions, and oils, as well as patches,adhesive plasters, bandages, dressings, depots, and reservoirs.

Tablets may be made by conventional means, e.g., compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the compound(prodrug) in a free-flowing form such as a powder or granules,optionally mixed with one or more binders (e.g., povidone, gelatin,acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g., lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, silica);disintegrants (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g., sodium lauryl sulfate);preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid); flavours, flavour enhancing agents, and sweeteners. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the compound (prodrug) thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with a coating, for example, to affect release, for example anenteric coating, to provide release in parts of the gut other than thestomach.

Ointments are typically prepared from the compound (prodrug) and aparaffinic or a water-miscible ointment base.

Creams are typically prepared from the compound (prodrug) and anoil-in-water cream base. If desired, the aqueous phase of the cream basemay include, for example, at least about 30% w/w of a polyhydricalcohol, i.e., an alcohol having two or more hydroxyl groups such aspropylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol andpolyethylene glycol and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe compound (prodrug) through the skin or other affected areas.Examples of such dermal penetration enhancers include dimethylsulfoxideand related analogues.

Emulsions are typically prepared from the compound (prodrug) and an oilyphase, which may optionally comprise merely an emulsifier (otherwiseknown as an emulgent), or it may comprises a mixture of at least oneemulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabiliser. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabiliser(s) make up the so-called emulsifying wax, and the waxtogether with the oil and/or fat make up the so-called emulsifyingointment base which forms the oily dispersed phase of the creamformulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulphate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the compound (prodrug) in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used.

Formulations suitable for intranasal administration, where the carrieris a liquid, include, for example, nasal spray, nasal drops, or byaerosol administration by nebuliser, include aqueous or oily solutionsof the compound (prodrug).

Formulations suitable for intranasal administration, where the carrieris a solid, include, for example, those presented as a coarse powderhaving a particle size, for example, in the range of about 20 to about500 microns which is administered in the manner in which snuff is taken,i.e., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose.

Formulations suitable for pulmonary administration (e.g., by inhalationor insufflation therapy) include those presented as an aerosol sprayfrom a pressurised pack, with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for ocular administration include eye dropswherein the compound (prodrug) is dissolved or suspended in a suitablecarrier, especially an aqueous solvent for the compound (prodrug).

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, natural orhardened oils, waxes, fats, semi-liquid or liquid polyols, for example,cocoa butter or a salicylate; or as a solution or suspension fortreatment by enema.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active compound, such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the compound(prodrug) is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the compound (prodrug) in the liquid is from about1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1μg/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the compounds (prodrugs), and compositions comprising thecompounds (prodrugs), can vary from patient to patient. Determining theoptimal dosage will generally involve the balancing of the level oftherapeutic benefit against any risk or deleterious side effects. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, the severity of the condition, and thespecies, sex, age, weight, condition, general health, and prior medicalhistory of the patient. The amount of compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the compound (prodrug) is in the range ofabout 0.1 to about 250 mg per kilogram body weight of the subject perday. Where the compound (prodrug) is a salt, a solvate, an ester, anamide, or the like, the amount administered is calculated on the basisof the parent compound and so the actual weight to be used is increasedproportionately.

Kits

One aspect of the invention pertains to a kit comprising (a) thecompound (prodrug), preferably provided in a suitable container and/orwith suitable packaging; and (b) instructions for use, for example,written instructions on how to administer the compound (prodrug), etc.

The written instructions may also include a list of indications forwhich the compound (prodrug) is a suitable treatment.

EXAMPLES

The following are examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

All starting materials, reagents and anhydrous solvents (eg. THF packedunder N₂) were purchased from Aldrich, unless otherwise stated.Kieselgel 60 (0.043–0.060) was used in gravity columns (Art 9385 and15111, Merck). TLC was performed on precoated sheets of Kieselgel 60F₂₅₄ (Art 5735, Merck). Melting points were determinated on a Koflerhot-stage (Reichert Thermovar) melting point apparatus and areuncorrected. Low resolution EI and FAB spectra were performed on aVG-2AB-SE double focusing magnetic sector mass spectrometer (FisonsInstruments, Warrington, Manchester, UK), operating at a resolution of1000.High resolution accurate mass spectra were determined on the samesystem, but with a resolution set to 8,000–10,000. Masses are measuredby peak matching the unknown with a mass of known composition. Reportedspectra are by FAB unless otherwise stated. NMR spectra were determinedin Me₂SO-d₆ on a Brucker AC250 spectrometer (250 MHz) at 30° C. (303 K)unless otherwise stated. IR spectra (film) were recorded on a PerkinElmer 1720X FT-IR spectrometer. Elemental analysis were determined byButterworth Laboratories Ltd. (Teddington, Middlesex, UK) and are within0.4% of theory except when stated. The chemical stability of theprodrugs and their propensity to behave as substrates for CPG2 weredetermined by HPLC.

Example 1 4-nitro-[bis(2′-hydroxyethyl)]-aniline (X-3a)

4-Nitrofluorobenzene (16.5 g, 11.7 mmol) was mixed with diethanolamine(35 mL) and the mixture was heated at 130° C. and stirred for 16 h. Thereaction mixture was cooled to 60° C., then poured into a beakercontaining NaOH (6 g) in water (1 L). The yellow precipitate wasrecovered by filtration and dried in dessicator for 24 h over P₂O₅, toafford the title compound (22 g, 83%) as a yellow solid.

¹H-NMR δ_(H) (ppm) 3.52–3.64 (m, 8H, N(CH₂, CH₂)₂OH), 4.82 (t, 2H, OH,J=5.29 Hz), 6.82 (d, 2H, H_(arom2+6), J=9.58 Hz), 8.01 (d, 2H,H_(arom3+5)).

Example 2 4-Nitro-bis[2′-(tert-butyldimethylsilyloxy)ethyl]-aniline(X-4a)

4-Nitro-[bis(2′-hydroxyethyl)]-aniline (X-3a) (5.0 g, 22.1 mmol) andtert-butyldimethylsilyl chloride (7.5 g, 50 mmol) were dissolved in 20mL DMF; imidazole (4.76 g, 70 mmol) was added, and the solution stirredat room temperature for 20 h. The solution was then concentrated andpurified by column chromatography (cyclohexane:AcOEt 1:1) to afford thetitle compound (8.9 g, 89%) as an yellow oil.

¹H-NMR δ_(H) (ppm): −0.03 (s, 12H, Si—CH₃), 0.81 (s, 18H, Si-t-Bu), 3.64(t, 4H, NCH₂, J=5.19 Hz), 3.78 (t, 4H, CH₂OSi), 6.83 (d, 2H,H_(arom2+6), J=9.37 Hz), 8.00 (d, 2H, H_(arom3+5)); MS m/z: 455 (M⁺+1,88), 477 (M⁺+23, 5), 439 (M⁺-Me, 53), 397 (M⁺−t-Bu, 30); acc. mass:(C₂₂H₄₃N₂O₄Si₂) calcd. 455.2761, found 455.2767. Anal. (C₂₂H₄₃N₂O₄Si₂)C, H, N.

Example 3 2-nitro-[bis(2′-hydroxyethyl)]-aniline (X-3b)

2-Nitrofluorobenzene (16.5 g, 11.7 mmol) was mixed with diethanolamine(35 mL) and the mixture was heated at 130° C. and stirred for 16 h. Thereaction mixture was cooled to 60° C., then poured into a beakercontaining NaOH (6 g) in water (1 L). The yellow precipitate wasrecovered by filtration and dried in dessicator for 24 h over P₂O₅, toafford the title compound as a yellow solid. After purification oncolumn (Kiselgel 60, 0.040–0.063, eluent: AcOEt) a yield of 61% (16.66g) was obtained.

¹H-NMR δ_(H) (ppm): 3.20 (t, 4H, —NHCH₂), 3.46 (t, 4H, CH₂OH), 4.54 (s,2H, OH), 6.64 (t, 1H, H_(arom4(5))), 7.39 (d, 1H, H_(arom3(6))), 7.49(t, 1H, H_(arom5(4))), 7.68 (d, 1H, H_(arom6(3))).

Example 4 2-Nitro-bis[2′-(tert-butyldimethylsilyloxy)ethyl]-aniline(X-4b)

The title compound was prepared using2-nitro-[bis(2′-hydroxyethyl)]-aniline (X-3b) in a method analogous tothat described in the previous Example. The product was purified bycolumn chromatography (eluent: cyclohexane:AcOEt 3:1) and obtained as ayellow oil (95%).

¹H-NMR δ_(H) (ppm): −0.03 (s, 12H, Si—CH₃), 0.80 (s, 18H, Si-t-Bu), 3.27(t, 4H, NCH₂, J=5.87 Hz), 3.65 (t, 4H, CH₂OSi), 6.98 (dt, 1H, H₅, J=7.58Hz), 7.37 (dd, 1H, H₃, J=8.43 Hz), 7.48 (dt, 1H, H₄, J=7.80 Hz), 7.67(dd, 1H, H₆, J=8.05 Hz); MS m/z: 455 (M⁺+1, 32), 397 (M⁺-t-Bu, 8), 309(M⁺-SiTBDM, 100); acc. mass: (C₂₂H₄₃N₂O₄Si₂) calcd. 455.2761, found455.2745. Anal. (C₂₂H₄₃N₄O₄Si₂) C, H; N required 6.17, found 6.94%.

Example 5 4-Amino-bis[2′-(tert-butyldimethylsilyloxy)ethyl]-aniline(X-5a)

4-Nitro-bis[2′-(tert-butyldimethylsilyloxy)ethyl]-aniline (X-4a) (5.50g, 12.1 mmol) was dissolved in 90 mL THF, 1.5 g Pd/C 10% was added, andthe suspension was stirred under H₂ atmosphere for 6 h. The catalyst wasthen filtered off, the solvent evaporated and the residue purified bycolumn chromatography (cyclohexane:AcOEt 3:1) to yield the titlecompound (4.90 g, 95%) as an oil.

¹H-NMR δ_(H) (ppm): 0.00 (s, 12H, Si—CH₃), 0.85 (s, 18H, Si-t-Bu), 3.30(t, 4H, NCH₂, J=6.20 Hz), 3.63 (t, 4H, CH₂OSi—), 4.34 (s, 2H, NH₂), 6.46(s, 4H, H_(arom)); MS m/z: 424 (M⁺, 70); acc. mass: (C₂₂H₄₄N₂O₂Si₂)calcd. 424.2941, found 424.2950. Anal. (C₂₂H₄₄N₂O₂Si₂): H, N; C required62.21, found 62.63%.

Example 6 2-Amino-bis[2′-(tert-butyldimethylsilyloxy)ethyl]-aniline(X-5b)

The title compound was prepared using 2-nitro-bis[2′-(tert-butyldimethylsilyloxy)ethyl]-aniline (X-4b) in a method analogous to that describedin the previous Example. The product was purified by columnchromatography (eluent: cyclohexane:AcOEt 3:1) and obtained as an oil(4.60 g, 99%).

¹H-NMR δ_(H) (ppm): 0.04 (s, 12H, Si—CH₃), 0.85 (s, 18H, Si-t-Bu), 3.02(t, 4H, NCH₂, J=6.19 Hz), 3.56 (t, 4H, CH₂OSi), 6.49 (dt, 1H, H₅, J=7.51Hz), 6.63 (dd, 1H, H₃, J=7.92 Hz), 6.78 (dt, 1H, H₄, J=7.54 Hz), 7.00(dd, 1H, H₆, J=7.81 Hz); MS m/z: 425 (M⁺+1, 28), 367 (M⁺-t-Bu+1, 15);acc. mass: (C₂₂H₄₅N₂O₂Si₂) calcd. 425.3020, found 425.3034.

Example 74-(N′-Benzyloxycarbonyl-amino)-N,N-bis[(2′-(tert-butyldimethylsilyl-oxy)ethyl]-aniline(X-6a)

4-Amino-N,N-bis[(2′-(tert-butyldimethylsilyl-oxy)ethyl]-aniline (X-5a)(3.74 g, 8.8 mmol) was dissolved in THF (100 mL) andN-(benzyloxycarbonyloxy)-succinimide (2.25 g, 9.0 mmol) was added. Thesolution was stirred at room temperature for 16 h. The solvent wasevaporated and the residue was purified by column chromatography(cyclohexane:ethyl acetate 1:1) to afford the title compound (4.65 g,95%) as an oil.

¹H-NMR δ_(H) (ppm): −0.01 (s, 12H, SiCH₃), 0.84 (s, 18H, Si-t-Bu), 3.41(t, 4H, NCH₂, J=5.86 Hz), 3.67 (t, 4H, CH₂OSi), 5.09 (s, 2H, PhCH₂),6.58 (d, 2H, H_(arom3+5), J=8.89 Hz), 6.82 (d, 2H, H_(arom2+6)),7.28–7.40 (m, 5H, H_(arom benzyl)), 9.28 (s, 1H, NH). MS m/z: 558 (M⁺,35), 423 (M⁺-PhCH₂—CO₂, 25); acc. mass: (C₃₀H₅₀N₂O₄Si₂) calcd. 558.3309,found 558.3330. Anal. (C₃₀H₅₀N₂O₄Si₂) C, H; N required 5.01, found4.60%.

Example 84-(N′-Benzyloxycarbonyl-N′-methyl-amino)-N,N-bis[(2′-(tert-butyidimethylsilyi-oxy)ethyl]-aniline(X-7a)

4-(N′-Benzyloxycarbonyl-amino)-N,N-bis[(2′-(tert-butyldimethylsilyl-oxy)ethyl]-aniline(X-6a) (5.2 g, 9.3 mmol) was dissolved in dry THF (60 mL) and NaH (60%in mineral oil, 0.6 g, 15 mmol) were added. After 40 min stirring atroom temperature under argon, methyl iodide (586 μL, 9.3 mmol) was addedand the stirring continued for 12 h. The solvent was evaporated, theresidue redissolved in ethyl acetate (100 mL) and extracted withdistilled water (100 mL). The organic layer was dried and evaporated toafford the title compound (5.34 g, 100%) as an oil.

¹H-NMR δ_(H) (ppm): 0.00 (s, 12H, Si—CH₃), 0.88 (s, 18H, Si-t-Bu), 3.21(s, 3H, N—CH₃), 3.45 (t, 4H, NCH₂, J=6.47 Hz), 3.71 (t, 4H, CH₂OSi),5.10 (s, 2H, PhCH₂), 6.60 (d, 2H, H_(arom3+5), J=9.08 Hz), 6.99 (d, 2H,H_(arom2+6), J=7.25 Hz), 7.20–7.35 (m, 5H, H_(arom benzyl)). MS m/z: 572(M⁺, 50), 595 (M⁺+Na, 7), 437 (M⁺-PhCH₂—CO₂, 45); acc. mass:(C₃₁H₅₂N₂O₄Si₂) calcd. 572.3466, found 572.3485.

Example 94-Methylamino-N,N-bis[(2′-(tert-butyldimethylsilyloxy)ethyl]-aniline(X-8a)

4-(N′-Benzyloxycarbonyl-N′-methyl-amino)-N,N-bis[(2′-(tert-butyldimethylsilyl-oxy)ethyl]-aniline(X-7a) (2.9 g, 5.06 mmol) was dissolved in ethyl acetate (120 mL), andPd/C 10% catalyst (1.6 g) added. The suspension was stirred under H₂atmosphere for 3 h. The catalyst was filtered off and the filtrate wasevaporated to afford the title compound (2.23 g, 100%) as an oil.

¹H-NMR δ_(H) (ppm): −0.01 (s, 12H, SiCH₃), 0.84 (s, 18H, Si-t-Bu), 2.58(s, 3H, N—CH₃), 3.22–3.32 (t, 4H, NCH₂), 3.63 (t, 4H, CH₂OSi, J=6.19Hz), 4.87 (s, 1H, NH), 5.10 (s, 2H, PhCH₂), 6.43 (d, 2H, H_(arom3+5),J=8.92 Hz), 6.65 (d, 2H, H_(arom2+6)), 7.20–7.35 (m, 5H,H_(arom benzyl)). MS m/z: 438 (M⁺, 100), 451 (M⁺+Na, 25); acc. mass:(C₂₃H₄₆N₂O₂Si₂) calcd. 438.3098, found 438.311500. Anal. (C₂₃H₄₆N₂O₂Si₂)C, H; N required 6.38, found 5.88.

Example 10 Diallyl L-glutamyl isocyanate (X-16)

To diallyl L-glutamate p-toluene sulfonate (the TsOH salt of diallylL-glutamate) (1.08 g, 2.7 mmol, NovaBiochem) and triphosgene (0.30 g,1.03 mmol) in 20 mL toluene, stirred at −78° C., trietylamine (0.86 mL,6.2 mmol) was added. After 30 min, the reaction mixture was allowed toreach room temperature and was used without further purification. Atypical IR spectrum was obtained v=2253 cm⁻¹ (NCO, v. intense).

Example 11 Diallyl N-{(4-{hydroxymethyl}phenyl)carbamoyl]-L-glutamate(X-18)

To a solution of diallyl glutamyl isocyanate (X-16) (25.0 mmol) in 100mL of THF were added 4-aminobenzyl alcohol (X-9) (3.0 g, 24.3 mmol,Lancaster) and triethylamine (3.41 mL, 24.3 mmol) in 20 mL of THF,dropwise, over 10 min, at room temperature. The reaction was completewithin 15 min. The reaction mixture was filtered and evaporated todryness; the residue was dissolved in 20 mL of EtOAc, washed with water(2×20 mL), and dried (MgSO₄), before evaporating again. A yellow oilresulted (10.25 g); 2.7 g of the obtained product was submitted topurification by preparative HPLC (CH₂Cl₂:EtOAc, 1:1) which yielded 1.52g (63%) of pure title compound.

v_(max) cm⁻¹ (film) 8354 (NH—, OH, broad), 1737 (C═O, ester), 1659 (C═O,urea); ¹H NMR δ_(H) 1.85–1.93 (m, 1H, CH₂CH(NH)—), 2.00–2.05 (m, 1H,—CH₂CH(NH)—), 2.46 (t, 2H, CH₂CO₂, J=5.4 Hz), 4.26–4.35 (m, 1H,—CH(NH)—), 4.40 (d, 2H, CH₂-Ph, J=5.6 Hz), 4.55 (d, 2H, CH₂O-allyl,J=5.3 Hz), 4.61 (d, 2H, CH₂O-allyl), 4.98 (t, 1H, OH), 5.17–5.37 (m, 4H,CH₂=allyl), 5.85–5.94 (m, 2H, CH=allyl), 6.56 (d, 1H, NH-G, J=8.0 Hz),7.17 (d, 2, H₂₊₆, J=8.5 Hz), 7.32 (d, 2H, H₃₊₅), 8.52 (s, 1H, NH-Ph); MBm/z 399 (M⁺+23, 35), 377 (M⁺+1, 100), 359 (M⁺-H₂O, 34). Anal.(C₁₉H₂₄N₂O₆) C, H, N.

Example 12 DiallylN-(4-{[4-nitrophenoxycarbonyloxy]methyl}phenyl-carbamoyl)-L-glutamate(X-19)

To a stirred solution of (X-18) (0.190 g, 0.50 mmol) in dry THF (10 mL)were added 4-nitrophenyl chloroformate (0.11 g, 0.5 mmol) andtriethylamine (0.1 mL, 0.6 mmol) at room temperature. The reaction wascomplete after 1 h. The formed precipitate was filtered and the solutionconcentrated under vacuum. AcOEt (10 mL) was added; the solution waswashed with brine (2×10 mL), dried (MgSO₄), and evaporated again, givingan oil which was purified by preparative HPLC to yield the titlecompound as a solid (0.132 g, 48.6%).

Mp 106–7° C.; v_(max) cm⁻¹ (film) 3356 (NH₂), 2933 (CH₂), 1766 (C=0,carbonate), 1738 (C═O, ester), 1660 (C═O, amide), 1525, 1346 (NO₂). ¹HNMR δ_(H) 1.86–1.95 (m, 1H, CH₂CH(NH)—), 2.00–2.05 (m, 1H, —CH₂CH(NH)—),2.46 (t, 2H, CH₂CO₂, J=5.4 Hz), 4.28–4.33 (m, 1H, —CH(NH)—), 4.55 (d,2H, CH₂O-allyl, J=5.3 Hz), 4.61 (d, 2H, CH₂O-allyl), 5.17–5.37 (m, 4H,CH₂=allyl), 5.22 (s, 2H, CH₂-Ph), 5.86–5.96 (m, 2H, CH=allyl), 6.65 (d,1H, NH-G, J=8.3 Hz), 7.34 (d, 2H, H₃₊₅, J=8.7 Hz), 7.43 (d, 2H, H₂₊₆),7.56 (d, 2H, H_(3′+5′), J=9.1 Hz, 8.31 (d, 2H, H_(2′+6′)), 8.71 (s, 1H,NH-Ph)(G=glutamic moiety; Ph=phenyl); Mass (C₂₈H₂₇N₃O₁₀Na) calcd,564.1594; found, 564.1590. Anal. (C₂₆H₂₇N₃O₁₀) H, N, C.

Example 13 Diallyl,4-{N-[4′-bis(2″-tert-butyldimethylsilyloxyethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-20a)

DiallylN-(4-{[4-nitrophenoxycarbonyloxy]methyl}phenyl-carbamoyl)-L-glutamate(X-19) (2.1 g, 3.9 mmol) and4-Methylamino-N,N-bis[(2′-(tert-butyldimethylsilyloxy)ethyl]-aniline(X-8a) (2.2 g, 5 mmol) were dissolved in DMA (50 mL) and stirred for 5days at room temperature. The solvent was evaporated and the residuepurified by column chromatography (CH₂Cl₂:AcOEt 9:1) to yield the titlecompound (0.92 g, 28%) as an oil.

¹H-NMR δ_(H) (ppm): 0.01 (s, 12H, Si—CH₃), 0.83 (s, 18H, Si-t-Bu),1.80–2.10 (2m, 2H, CH₂CH(NH)—), 2.44 (t, 2H, CH₂CO₂, J=8.25 Hz), 3.11(s, 3H, N—CH₃), 3.46 (t, 4H, NCH₂, J=5.58 Hz), 3.69 (t, 4H, CH₂OSi—),4.25–4.35 (m, 1H, CH(NH)CH₂), 4.53 (d, 2H, CH₂O allyl, J=5.45 Hz), 4.59(d, 2H, CH₂O allyl, J=6.38 Hz), 4.94 (s, 2H, PhCH₂), 5.14–5.38 (m, 4H,CH₂=allyl), 5.80–6.00 (m, 2H, CH=allyl), 6.60 (d, 3H,H_(arom3′+5═)+NH-G, J=8.70 Hz), 6.99 (d, 2H, H_(arom2′+6′)), 7.15 (d,2H, H_(arom2+6)), 7.32 (d, 2H, H_(arom3+5), J=8.20 Hz), 8.60 (s, 1H,PhNH); MS m/z: 841 (M⁺+1, 5), 864 (M⁺+Na, 3), 437 (M⁺-L1ACH₂OCO, 100);acc. mass: (C₄₃H₆₉N₄O₉Si₂) calcd. 841.4603, found 841.4630. Anal.(C₄₃H₆₈N₄O₉Si₂): C, H; N required 6.66, found 6.22.

Example 14 Diallyl,4-{N-[4′-bis(2″-hydroxyethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-21a)

Diallyl,4-{N-[4′-bis(2″-tert-butyldimethylsilyloxyethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-20a) (0.85 g, 1.0 mmol) was dissolved in 25 mL THF, 2.5 mLtriethylamine trihydrofluoride were added and the solution stirred atroom temperature for 7 h. The solvent was evaporated, the residue wasdiluted with AcOEt and extracted with H₂O (100 mL), saturated aqueousNaHCO₃ (200 mL), again with H₂O (100 mL), dried (MgSO₄) and evaporatedto yield the title compound (0.58 g, 93.6%) as a gum.

¹H-NMR δ_(H) (ppm): 1.75–2.15 (2m, 2H, CH₂CH(NH)—), 2.44 (t, 2H, CH₂CO₂,J=8.54 Hz), 3.12 (s, 3H, N—CH₃), 3.38 (t, 4H, NCH₂, J=5.50 Hz), 3.50 (t,4H, CH₂OH), 4,25–4.35 (m, 1H, CH(NH)CH₂), 4.53 (d, 2H, CH₂O allyl,J=5.31 Hz), 4.59 (d, 2H, CH₂O allyl, J=5.28 Hz), 4.71 (t, 2H, OH, J=5.45Hz), 4.94 (s, 2H, PhCH₂), 5.15–5.38 (m, 4H, CH₂=allyl), 5.85–6.00 (m,2H, CH=allyl), 6.61 (d, 3H, H_(arom3′+5′)+NH-G, J=8.90 Hz), 6.99 (d, 2H,H_(arom2′+6′)), 7.17 (d, 4H, H_(arom2+6)), 7.33 (d, 2H, H_(arom3+5),J=8.09 Hz), 8.62 (s, 1H, PhNH); MS, m/z: 613 (M⁺+1, 45); acc. mass:(C₃₁H₄₁N₄O₉) calcd. 613.2874, found 613.2853. Anal. (C₃₁H₄₀N₄O₉): C, H,N.

Example 15 Diallyl,4-{N-[4′-bis(2″-mesyloxyethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-22a)

Over a solution of diallyl,4-{N-[4′-bis(2″-hydroxyethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-21a) (0.58 g, 0.95 mmol), 4-N-dimethylaminopyridine (25 mg, 0.2 mmol)and NEt₃ (420 μL, 3.0 mmol) in CH₂Cl₂ (10 mL), mesyl anhydride (0.487 g,2.8 mmol) dissolved in CH₂Cl₂ (15 mL) was added. After stirring at roomtemperature for 2.5 h, the solution was diluted with CH₂Cl₂ to 50 mL,extracted with 10% aq. citric acid (2×50 mL), aq. NaHCO₃ (50 mL),distilled water (50 mL), dried over MgSO₄ and evaporated to afford thetitle compound (0.73 g, 100%) as a gum.

¹H-NMR δ_(H) (ppm): 1.85–2.20 (2m, 2H, CH₂CH(NH)—), 2.44 (t, 2H, CH₂CO₂,J=7.77 Hz), 3.14 (s, 9H, CH₃SO₃, N—CH₃), 3.71 (t, 4H, NCH₂), 4.29 (t,5H, CH₂OMes+CH(NH)CH₂, J=4.95 Hz), 4.54 (d, 2H, CH₂O allyl, J=5.40 Hz),4.60 (d, 2H, CH₂O allyl, J=5.30 Hz), 4.96 (s, 2H, PhCH₂), 5.15–5.37 (m,4H, CH₂=allyl), 5.80–6.00 (m, 2H, CH=allyl), 6.61 (d, 1H, NH-G, J=7.99Hz), 6.75 (d, 2H, H_(arom3′+5′), J=9.00 Hz), 7.08 (d, 2H,H_(arom2′+6′)), 7.18 (d, 2H, H_(arom2+6)) 7.33 (d, 2H, H_(arom3+5),J=8.24 Hz), 8.61 (s, 1H, PhNH). MS m/z: 769 (M⁺+1, 4); acc. mass:(C₃₃H₄₅N₄O₁₃S2) calcd. 769.2425, found 769.2456. Anal. (C₃₃H₄₄N₄O₁₃S₂):C, H, N, S.

Example 16 Diallyl,4-{N-[4′-bis(2″-iodoethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-23a-I)

A solution of diallyl,4-{N-[4′-bis(2″-mesyloxyethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-22a) (0.25 g, 0.33 mmol) and NaI (0.75 g, 5.0 mmol) in 25 mL acetonewas stirred at reflux for 4 hrs. The solvent was evaporated, the residueretaken in 30 mL AcOEt and washed with 20 mL H₂O, dried (MgSO₄) andevaporated. The residue was purified by column chromatography(cyclohexane:AcOEt 1:1) to afford the title compound (0.165 g, 60%), asa solid, mp 138–140° C.

¹H-NMR δ_(H) (ppm): 1.80–2.15 (2m, 2H, CH₂CH(NH)—), 2.44 (t, 2H,CH₂CO₂), 3.13 (s, 3H, N—CH₃), 3.25–3.35 (t, 4H, NCH₂), 3.70 (t, 4H,CH₂I, J=6.17 Hz), 4.25–4.40 (m, 1H, CH(NH)CH₂), 4.54 (d, 2H, CH₂O allyl,J=6.27 Hz), 4.60 (d, 2H, CH₂O allyl, J=4.42 Hz), 4.95 (s, 2H, PhCH₂),5.10–5.37 (m, 4H, CH₂=allyl), 5.80–6.00 (m, 2H, CH=allyl), 6.62 (d, 3H,H_(arom3′+5′)+NH-G, J=8.67 Hz), 7.09 (d, 2H, H_(arom2′+6′)), 7.18 (d,2H, H_(arom2+6)), 7.33 (d, 2H, H_(arom3+5), J=7.72 Hz), 8.61 (s, 1H,PhNH). MS m/z: 833 (M⁺+1, 7), 855 (M⁺+Na, 14); acc. mass:(C₃₁H₃₈N₄O₇I₂Na) calcd. 855.0728, found 855.0755. Anal. (C₃₁H₃₈N₄O₇I₂):H, N; C required 44.73, found 45.24%.

Example 17 Diallyl,4-{N-[4′-bis(2″-bromoethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-23a-Br)

To a THF solution (25 mL) of diallyl,4-{N-[4′-bis(2″-mesyloxyethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-22a) (0.26 g, 0.34 mmol), LiBr (0.44 g, 5.0 mmol) was added. After1.5 h stirring at reflux the solvent was evaporated, the residue retakenin CH₂Cl₂ (25 mL), extracted with H₂O (25 mL), the organic layer dried(MgSO₄) and evaporated to dryness. Purification was achieved bypreparative HPLC (cyclohexane:AcOEt 3:1), and the title compound (0.16g, 64%) was obtained as a solid, mp 104–107° C.

¹H-NMR δ_(H) (ppm): 1.80–2.15 (2m, 2H, CH₂CH(NH)—), 2.44 (t, 2H,CH₂CO₂), 3.13 (s, 3H, N—CH₃), 3.56 (t, 4H, NCH₂, J=6.74 Hz), 3.75 (t,4H, CH₂Br), 4.25–4.35 (m, 1H, CH(NH)CH₂), 4.54 (d, 2H, CH₂O allyl,J=5.52 Hz), 4.59 (d, 2H, CH₂O allyl, J=5.21 Hz), 4.95 (s, 2H, PhCH₂),5.10–5.37 (m, 4H, CH₂=allyl), 5.80–6.00 (m, 2H, CH=allyl), 6.61 (d, 1H,NH-G, J=8.34 Hz), 6.68 (d, 2H, H_(arom3′+5′), J=8.89 Hz), 7.09 (d, 2H,H_(arom2′+6′)), 7.18 (d, 2H, H_(arom2+6)), 7.33 (d, 2H, H_(arom3+5),J=8.26 Hz), 8.61 (s, 1H, PhNH). MS m/z: 738 (M⁺, 5), 761 (M⁺+Na, 15);acc. mass: (C₃₁H₃₈N₄O₇Br₂) calcd. 759.1005, found 759.1021. Anal.(C₃₁H₃₈N₄O₇Br₂): C, H, N.

Example 18 Diallyl,4-{N-[4′-bis(2″-chloroethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-23a-Cl)

Diallyl,4-{N-[4′-bis(2″-mesyloxyethyl)amino-phenyl]-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-22a) (0.245 g, 0.32 mmol) was dissolved in DMA (25 mL). Lithiumchloride (0.21 g, 5 mmol) was added to the reaction mixture and it wasstirred for 24 h at room temperature. The solvent was evaporated, theresidue taken in AcOEt (50 mL) and extracted with distilled water (2×50mL). The organic layer was separated, dried (MgSO₄) and evaporated. Theresidue was purified by column chromatography (eluent AcOEt:cyclohexane1:1) to afford the title compound (0.105 g, 51%) as a gum.

¹H-NMR δ_(H) (ppm): 1.80–2.20 (2m, 2H, CH₂CH(NH)—), 2.44 (t, 2H, CH₂CO₂,J=8.26 Hz), 3.13 (s, 3H, N—CH₃), 3.70 (s, 8H, N(CH₂CH₂Cl)₂), 4.25–4.40(m, 1H, CH(NH)CH₂), 4.54 (d, 2H, CH₂O allyl, J=5.44 Hz), 4.59 (d, 2H,CH₂O allyl, J=5.35 Hz), 4.95 (s, 2H, PhCH₂), 5.10–5.35 (m, 4H,CH₂=allyl), 5.80–6.00 (m, 2H, CH=allyl), 6.61 (d, 1H, NH-G, J=8.35 Hz),6.70 (d, 2H, H_(arom3′+5′), J=8.86 Hz), 7.08 (d, 2H, H_(arom2′+6′)),7.18 (d, 2H, H_(arom2+6)), 7.33 (d, 2H, H_(arom3+5), J=8.56 Hz), 8.61(s, 1H, PhNH). MS m/z: 671 (M⁺+Na, 20); acc. mass: (C₃₁H₃₈N₄O₇Cl₂Na)calcd. 671.2015, found 671.2037. Anal. (C₃₁H₃₈N₄O₇Cl₂): C, H, N Cl.

Example 194-{N-[4′-bis(2″-iodoethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamicacid (X-24a-I) (P-1)

Diallyl,4-{N-[4′-bis(2″-iodoethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-23a-I) (0.145 g, 0.17 mmol) and Pd tetrakis-triphenylphosphine (15mg, 15 μmol) were dissolved in 4 mL CH₂Cl₂. Pyrrolidine (58 μL, 0.70mmol) was added in one portion. After 30 min stirring, the solution wasdiluted with AcOEt. The pyrrolidine salt of the deprotected carboxylicacid precipitated at once. The reaction mixture was partiallyevaporated, the remaining solvent was diluted with AcOEt andconcentrated to remove CH₂Cl₂ selectively. The precipitate left in theflask after discarding the solvent was washed twice with AcOEt, dried,dissolved in 8 mL methanol and eluted through a column loaded with 40cm³ IRC50 resin (H form) previously washed with MeOH. After evaporationthe elute yield the title compound as a solid (0.105 g, 77.8%), mp103–105° C.

¹H-NMR δ_(H) (ppm): 1.75–2.00 (2m, 2H, CH₂CH(NH)—), 2.28 (t, 2H, CH₂CO₂,J=7.43 Hz), 3.14 (s, 3H, N—CH₃), 3.30 (t, 4H, NCH₂), 3.72 (t, 4H, CH₂I,J=7.64 Hz), 4.10–4.25 (m, 1H, CH(NH)CH₂), 4.96 (s, 2H, PhCH₂), 6.46 (d,1H, NH-G, J=7.48 Hz), 6.64 (d, 2H, H_(arom3′+5′), J=8.85 Hz), 7.10 (d,2H, H_(arom2′+6′)), 7.17 (d, 2H, H_(arom2+6), J=7.49 Hz), 7.34 (d, 2H,H_(arom3+5)), 8.68 (s, 1H, PhNH). MS m/z: 752 (M⁺+1, 10), 775 (M⁺+Na,35); acc. mass: (C₂₅H₃₀N₄O₇I₂Na) calcd. 775.0102, found 775.0088.

Example 204-{N-[4′-bis(2″-bromoethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamicacid (X-24a-Br) (P-2)

The title compound was prepared from diallyl,4-{N-[4′-bis(2″-bromoethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-23a-Br) by a method analogous to that described in the previousExample (0.10 g, 80%), mp 75–77° C.

¹H-NMR δ_(H) (ppm): 1.80–1.95 (m, 2H, CH₂CH(NH)—), 2.28 (t, 2H, CH₂CO₂,J=7.33 Hz), 3.14 (s, 3H, N—CH₃), 3.71 (s, 8H, N(CH₂CH₂Cl)₂), 4.10–4.20(m, 1H, CH(NH)CH₂), 4.95 (s, 2H, PhCH₂), 6.48 (d, 1H, NH-G, J=7.17 Hz),6.68 (d, 2H, H_(arom3′+5′), J=8.74 Hz), 7.09 (d, 2H, H_(arom2′+6′)),7.18 (d, 2H, H_(arom2+6)), 7.34 (d, 2H, H_(arom 3+5), J=8.45 Hz), 8.74(s, 1H, PhNH). MS m/z: 658 (M⁺, 8), 681 (M⁺+Na, 15); acc. mass:(C₂₅H₃₀N₄O₇Br₂Na) calcd. 679.0379, found 679.0386.

Example 214-{N-[4′-bis(2″-chloroethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamicacid (X-24a-Cl) (P-3)

The title compound was prepared from diallyl,4-{N-[4′-bis(2″-chloroethyl)amino-phenyl]-N-methyl-carbamoyl-oxymethyl}-phenyl-carbamoyl-L-glutamate(X-23a-Cl) by a method analogous to that described in the previousExample (0.075 g, 90%), mp 70–72° C.

¹H-NMR δ_(H) (ppm): 1.75–2.00 (2m, 2H, CH₂CH(NH)—), 2.27 (t, 2H,CH₂CO₂), 3.14 (s, 3H, N—CH₃), 3.71 (s, 8H, N(CH₂CH₂Cl)₂), 4.10–4.20 (m,1H, CH(NH)CH₂), 4.95 (s, 2H, PhCH₂), 6.49 (d, 1H, NH-G), 6.70 (d, 2H,H_(arom3′+5′), J=8.60 Hz), 7.09 (d, 2H, H_(arom2′+6′)), 7.18 (d, 2H,H_(arom2+6)), 7.34 (d, 2H, H_(arom3+5), J=8.14 Hz), 8.71 (s, 1H, PhNH).MS m/z: 568 (M⁺, 25), 591 (M⁺+Na, 100); acc. mass: (C₂₅H₃₀N₄O₇Cl₂Na)calcd. 591.1389, found 591.1371.

Biological Data

Cytotoxicity Assays

Prodrugs of the invention (P), comparison prodrugs (CP), and some of thecorresponding drugs (D) were tested for cytotoxicity in WiDr cells (acolon carcinoma cell line) engineered for stable expression of(stCPG2(Q)3) or, as a control, the non-prodrug activating enzymeβ-galactosidase (β-gal). The construction of WiDr cells engineered tostably express stCPG2(Q)3 or β-gal, was performed as previouslydescribed for other cell lines (see Marais et al., 1997; Niculescu-Duvazet al., 1998b).

Cells (2×10⁶) were seeded into 6-well plates, producing confluentmonolayers in 48 hrs. Compounds were dissolved in DMSO at 10 mM (CP-2,CP-4, CP-9, CP-11), 20 mM (D-1, D-3, D-5, D-6), or 50 mM (all others),immediately prior to treatment, diluted in culture medium, and added tothe wells. A similar concentration of compound solution was added afteran incubation of 1 hour, and the cells were incubated for an additional20 hrs. The cells were harvested and re-seeded in quadruplicate in96-well plates at ˜2×10³/well and incubated until the control wellsachieved confluence. The plates were then fixed and stained withsulforhodamine-B, the extinction at 590 nm was determined, and theresults expressed as percentage of control growth as a function oflog(dose). The IC₅₀ was determined by non-linear regression to a logdose-effect sigmoid, constraining the minimum to be positive (usingGraphPad Prism®, GraphPad Software Inc., San Diego, Calif., USA).

For comparison purposes, the prodrugN-{4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl}-L-glutamic acidprodrug (CMDA, see below), which has undergone clinical trials in ADEPT(see Martin et al., 1997; Napier et al., 2000), was also tested.

The degree of activation is defined as the ratio of the IC₅₀ value ofthe prodrug in the β-gal expressing cell line to the IC₅₀ value of theprodrug in CPG2 expressing cell line.

The results are summarised in the following tables, wherein the prodrugsand drugs have the following formulae:

TABLE 1 Cytotoxicity Data (Prodrugs)

IC₅₀(μM) Cmpd. Z o/p R^(N) X¹ X² LacZ stCPG2(Q)3 Degree of activation(fold) CMDA n/a para H Cl Cl 3230 [±120] 100 [±10] 32.3 CP-1 NH ortho HI I 149.0 3.0 49.7 (80.7–275.0) (2.1–4.4) CP-2 NH para H I I 179.8 1.8101.6 (71.7–449.4) (1.0–3.2) CP-3 NH ortho H Br Br 117.7 4.7 24.9(83.8–165.2) (7.8–27.8) CP-4 NH para H Br Br 204.6 3.1 66.6(108.6–384.6) (1.8–5.3) CP-5 NH ortho H Cl Cl 21.1 4.5 4.7 (12.0–36.9)(2.9–7.1) CP-6 NH para H Cl Cl — — — CP-7 O ortho H OMes OMes 208.2 40.05.2 (86.4–500.7) (22.7–70.3) CP-8 O ortho H I I 73.3 19.0 3.9(45.3–119.8) (11.7–30.9) CP-9 O para H I I 53.6 1.3 42.5 (36.9–78.0)(0.8–2.0) CP-10 O ortho H Br Br 183.4 95.2 1.9 (83.8–400.4) (57.1–159)CP-11 O para H Br Br 99.7 2.6 38.3 (19.1–520.6) (1.2–6.0) CP-12 O orthoH Cl Cl 38.1 28.1 1.4 (19.1–76) (11.1–43.6) CP-13 O para H Cl Cl — — —CP-14 O ortho H OMes Cl 148.4 28.5 5.2 (65.1–338.2) (17.5–47.5) CP-15 Opara Me I I 21.5 2.6 8.3 (16.2–28.4) (1.7–4.1) CP-16 O para Me Br Br27.2 3.6 7.5 (14.1–52.5) (2.2–6.0) CP-17 O para Me Cl Cl 19.6 1.7 11.5(15.0–25.7) (1.1–2.5) P-1 NH para Me I I 184.7 1.5 124.0 (105.4–324.4)(0.8–2.6) P-2 NH para Me Br Br 332.4 4.1 81.4 (222.6–496.3) (2.5–6.7)P-3 NH para Me Cl Cl 39.7 2.5 15.9 (27.0–58.4) (1.6–3.9) Note: the round( ) bracketed values indicate the 95% confidence intervals. Note: ForCMDA, the square [ ] bracketed values represent the standard errors ofthe mean as published in Spooner et al., 2000.

TABLE 2 Cytotoxicity Data (Drugs) Degree of Cmpd. IC₅₀(μM) activation noo/p R^(N) X¹ X² LacZ stCPG2(Q)3 (fold) D-1 para H I I 10.2 8.6 1.2(4.8–18.3) (4.2–14.0) D-2 ortho H Br Br 23.4 18.6 1.3 (12.4–44.1)(10.7–32.2) D-3 para H Br Br 9.4 7.7 1.2 (5.7–18.5) (5.0–14.8) D-4 orthoH Cl Cl 3.5 2.4 1.5 (2.1–5.7) (1.3–4.4) D-5 para Me I I 7.7 8.4 0.92(4.1–14.5) (4.6–15.5) D-6 para Me Br Br 7.2 7.4 0.97 (3.6–14.4)(3.5–15.5) D-7 para Me Cl Cl 9.2 8.2 1.1 (5.7–15.1) (5.3–12.5) Note: thebracketed ( ) values indicate the 95% confidence intervals.

The differential obtained in tumor cells transfected with CPG2 appearsto be dependent upon an optimal chemical reactivity of the prodrugs andthe corresponding drugs. This is in good agreement with previousobservation on the in vitro and in vivo behaviour of the direct prodrugsin CPG2-based GDEPT systems (see Friedlos et al., 2002). The increasedlipophilicity of these prodrugs and drugs could also be important fortheir improved biological activity.

The inventors postulate that N-alkylation (e.g., N-methylation) of theprodrug results in (a) improved stability of the carbamate linkagebetween the linker and the drug moiety, and (b) increased basicity, andtherefore increased chemical reactivity, of the released nitrogenmustard drug.

N-alkylation (e.g., N-methylation) has a beneficial effect on theirbiological activity despite no observed benefit to the kinetics. Theprodrugs show lower chemical reactivity than the non-methylatedcounterparts, presumably due to increased stability of the secondarycarbamate. This effect is minimal at the drug level.

As a general observation, prodrugs incorporating ureas (Z=NH) are moreeffective than those containing carbamates (Z=O). The five mosteffective prodrugs, in terms of differential, are all ureas (Z=NH).

The use of I and Br instead of Cl as leaving groups in the nitrogenmustards leads to drugs with shorter half-lives and increased potency(IC₅₀=0.5–2.7 μM). The corresponding prodrugs also exhibited shorterhalf-lives. Nonetheless, for many of the prodrugs the differentials inthe WiDr cell lines are better than those of CMDA (CP-1, CP-2, CP-4,CP-9, CP-11, P-1, P-2). The two most effective prodrugs in terms ofdifferential are the iodo derivatives CP-2 and P-1. The most effectiveprodrug in terms of differential is the N-methylated iodo derivativeP-1.

The prodrugs belonging to the ortho series are less effective than theirpara counterparts. However, even in the ortho series, the I and Brnitrogen mustard prodrugs are the most active and significantdifferentials were obtained in the transfected WiDr cell line (50 and 25fold for CP-1 and CP-3 respectively). The ortho prodrugs had lowerK_(m)'s with respect to the linkers and the para series, which is ofpotential benefit in in vivo situations.

Aqueous Half-Life Determination

The chemical half-lives of the prodrugs and some of the correspondingdrugs were determined by HPLC or a spectrophotometric method.

Compounds were prepared as 10 mM concentrates in MeOH (D-2, D-4) or inDMSO (all others) and diluted 100 fold in CPG2 assay buffer (100 mMTris-HCl, pH 7.3; 260 □M ZnCl₂; 1 mL) to give 100 μM solutions. Aliquots(10 μL) were injected into a Partisphere C18 column (125×4.6 mm, 5 μm,Whatman) (compounds D4, D-7) or a Synergi Polar RP phenyl phase column(150×4.6 mm, 4 μm, Phenomenex) (all others) and eluted isocratically (1mL/min) with 10 mM ammonium acetate (pH 5.0) containing percentages ofmethanol (65–85%) that gave retention times of 3–4 minutes. The eluatewas monitored at 265–275 nm (CP-9, CP-11, CP-15, CP-16, CP-17) or 250 nm(all others). The amount of starting material remaining after variousperiods of incubation was determined either by repeat injection from asingle vial (CP-5, CP-12, CP-15, CP-17, P-1, P-3), or by delayedinjections from a new vial each time (all others). The results wereexpressed as fraction of starting material as a function of time, andthe half-life determined by non-linear regression to a one-phaseexponential decay, constraining the maximum to 1 and the minimum to 0(GraphPad Prism®).

Compounds D-1, D-2, D-3, D-5 and D-6 proved too labile for half-lifedetermination by HPLC, and a spectrophotometric method was employed. Thechange in absorbance on dilution into aqueous conditions at a wavelengthpreviously determined to give the largest difference was monitored for 3min at a sampling rate of 100/min. The data were fitted to a rising orfalling exponential by linear regression with no constraints (GraphPadPrism®), and the half life calculated as 0.69/rate constant. It wasestablished that this method gave similar results to the moreunequivocal HPLC method.

The results are summarised in the following tables.

TABLE 3 Aqueous Half Lives (Prodrugs) T1/2 Comp prodrug T1/2 prodrug/No. Z o/p R^(N) X¹ X² (min) T1/2 drug CP-1 NH ortho H I I 2.7 — CP-2 NHpara H I I 0.97 1.9 CP-3 NH ortho H Br Br 3.0 3.5 CP-4 NH para H Br Br0.85 0.4 CP-5 NH ortho H Cl Cl 107.5 13.9 CP-6 NH para H Cl Cl CP-7 Oortho H OMes OMes 10.8 — CP-8 O ortho H I I 2.1 — CP-9 O para H I I 0.982.0 CP-10 O ortho H Br Br 3.47 3.5 CP-11 O para H Br Br 0.9 0.4 CP-12 Oortho H Cl Cl 146.3 11.9 CP-13 O para H Cl Cl CP-14 O ortho H OMes Cl10.0 — CP-15 O para Me I I 4.6 7.7 CP-16 O para Me Br Br 2.7 1.0 CP-17 Opara Me Cl Cl 173.6 20.6 P-1 NH para Me I I 3.9 6.5 P-2 NH para Me Br Br2.8 1.0 P-3 NH para Me Cl Cl 173.6 20.9

TABLE 4 Aqueous Half-lives (Drugs) T1/2 Compd. no o/p R^(N) X¹ X² (min)D-1 para H I I 0.5 D-2 ortho H Br Br 1.0 D-3 para H Br Br 2.3 D-4 orthoH Cl Cl 12.3 D-5 para Me I I 0.6 D-6 para Me Br Br 2.7 D-7 para Me Cl Cl8.3

Difficulties were encountered in determining the half-lives of the orthonitrogen mustards. All the ortho nitrogen mustard drugs (D-1, D-2, andD-4) have the correct microanalysis. However ¹H-NMR (in DMSO-d₆) andLC-MS (in DMSO-buffer) showed rapid cyclisation to the correspondingbenzopiperidine derivative (as shown below), except for thebis(chloroethyl) derivative D-4.

Comparisons based upon 4-amino (R^(N)═H) versus 4-methylamino(R^(N)=Me), and 4-amino (para) versus 2-amino (ortho) are summarised inthe following tables.

TABLE 5 Comparison of Half-Lives: 4-amino (R^(N) = H) versus4-methylamino (R^(N) = Me) Comps. Z o/p X¹ X² T_(1/2) ratio CP-6/P-3 NHpara Cl Cl 0.28 CP-4/P-2 NH para Br Br 0.30 CP-2/P-1 NH para I I 0.25CP-13/CP-15 O para Cl Cl 0.30 CP-10/CP-14 O para Br Br 0.33 CP-8/CP-13 Opara I I 0.21

TABLE 6 Comparison of Half-Lives: 4-amino (para) versus 2-amino (ortho)Comps. Z R^(N) X¹ X² T_(1/2) ratio CP-6/CP-5 NH H Cl Cl 0.45 CP-4/CP-3NH H Br Br 0.28 CP-2/CP-1 NH H I I 0.36 CP-13/CP-12 O H Cl Cl 0.35CP-11/CP-10 O H Br Br 0.26 CP-9/CP-8 O H I I 0.47

In general, the 4-amino aniline mustard prodrugs have shorter half livesthan the N-methylated and their 2-amino counterparts.

The half-lives of the N-methylated prodrugs are consistently 3–4 timeslonger than the corresponding non-methylated prodrugs, irrespective ofthe halogen in the mustard moiety or the type of linker (Z=NH or Z=O).

Similarly the ortho amino analogues have half-lives 2–3 times longerthan para amino prodrugs.

Enzyme Kinetics

The kinetics of activation of the self-immolative prodrugs by CPG2 wasmeasured for the bis(chloroethyl) series. This series was chosen inorder to minimise the influence of the nitrogen mustard moietyhydrolysis on the measured kinetics.

Reactions were set up containing CPG2 assay buffer (1 mL), CPG2 (50 mU)and prodrug (5–50 μM in steps of 5 μM) from concentrates as above. Thevials were incubated at 37° C., and the amount of prodrug remaining inthe mixture was determined by HPLC as above at 0, 5, 10, 15 and 20minutes post start. The rate of loss of compound in μM/min wasdetermined by regression. The rate of chemical-only loss, calculatedfrom the first derivative of the equation for exponential decay, wassubstracted, and the kinetic parameters derived from non-linearregression to the Michaelis-Menten equation (GraphPad Prism®).

The results are summarised in the following table.

TABLE 7 Kinetics Results K_(m) k_(cat) K_(cat)/K Compd. no Z o/p R^(N)X¹ X² (□M) (s⁻¹) m L1-OH^((a)) NH n/a n/a n/a n/a 3.1 65.4 21.1L2-OH^((b)) O n/a n/a n/a n/a 1.7 140 82.3 CP-5 NH ortho H Cl Cl 0.552.92 5.31 CP-6 NH para H Cl Cl <5 10–50 — CP-12 O ortho H Cl Cl 1.034.27 4.14 CP-13 O para H Cl Cl <5 <10 — CP-17 O para Me Cl Cl 2.43 4.321.78 P-3 NH para Me Cl Cl 6.05 5.50 0.91

Two analogs, (a) and (b), were included for comparison purposes. Theyare:

-   (c) L1-OH═N-[4-(hydroxymethyl)phenyl-carbamoyl]-L-glutamatic acid.-   (d) L2-OH═N-[4-(hydroxymethyl)phenyl-oxycarbonyl]-L-glutamatic acid.

All prodrugs showed K_(m)'s comparable with those of L1-OH (Z=NH) andL2-OH (Z=O) (see Niculescu-Duvaz et al., 1998b). This indicates a goodstructural fit for the CPG2 active site, even with the orthoderivatives. However, the k_(cat) remains low compared to the directprodrugs and the linkers alone.

The foregoing has described the principles, preferred embodiments, andmodes of operation of the present invention. However, the inventionshould not be construed as limited to the particular embodimentsdiscussed. Instead, the above-described embodiments should be regardedas illustrative rather than restrictive, and it should be appreciatedthat variations may be made in those embodiments by workers skilled inthe art without departing from the scope of the present invention asdefined by the appended claims.

REFERENCES

A number of patents and publications are cited above in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Full citations for these references areprovided below. Each of these references is incorporated herein byreference in its entirety into the present disclosure, to the sameextent as if each individual reference was specifically and individuallyindicated to be incorporated by reference.

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1. A compound of the formula:

wherein: R^(N) is independently C₁₋₇alkyl; X¹ is independently —I, —Br, or —Cl; X² is independently —I, —Br, or —Cl; the group —N(CH₂CH₂X¹)(CH₂CH₂X²) is independently attached at the 2-position or at the 4-position; each R^(G) is independently —H or an ester substituent; n is independently an integer from 0 to 4; each R^(P), if present, is independently a phenyl substituent; m is independently an integer from 0 to 4; each R^(M), if present, is independently a mustard substituent; and pharmaceutically acceptable salts, solvates, amides, and esters thereof.
 2. A compound according to claim 1, wherein R^(N) is independently unsubstituted al iphatic C₁₋₇alkyl.
 3. A compound according to claim 1, wherein R^(N) is independently unsubstituted al iphatic C₁₋₄alkyl.
 4. A compound according to claim 1, wherein R^(N) is independently -Me, -Et, -nPr, -iPr, -allyl, -nBu, -sBu, -iBu, or -tBu.
 5. A compound according to claim 1, wherein R^(N) is independently -Me or -Ft.
 6. A compound according to claim 1, wherein R^(N) is independently -Me.
 7. A compound according to claim 1, wherein each of X¹ and X² is independently —I.
 8. A compound according to claim 1, wherein each of X¹ and X² is independently —Br.
 9. A compound according to claim 1, wherein each of X¹ and X² is independently —Cl.
 10. A compound according to claim 1, wherein R^(N) is independently C₁₋₄alkyl; and, each X is independently —Cl, —Br or —I.
 11. A compound according to claim 1, wherein R^(N) is independently -Me; and, each X is independently —Cl, —Br or —I.
 12. A compound according to claim 1, wherein R^(N) is independently C₁₋₄alkyl; and, each X is independently —I.
 13. A compound according to claim 1, wherein R^(N) is independently -Et or -Me; and, each X is independently —I.
 14. A compound according to claim 1, wherein R^(N) is independently -Me; and, each X is independently —I.
 15. A compound according to claim 1, wherein the group —N(CH₂CH₂X¹)(CH₂CH₂X²) is independently attached at the 4-position.
 16. A compound according to claim 1, wherein R^(N) is independently C₁₋₄alkyl; each X is independently —Cl, —Br or —I; and, the group —N(CH₂CH₂X)₂ is independently attached at the 4-position.
 17. A compound according to claim 1, wherein R^(N) is independently -Me; each X is independently —Cl, —Br or —I; and, the group —N(CH₂CH₂X)₂ is independently attached at the 4-position.
 18. A compound according to claim 1, wherein R^(N) is independently C₁₋₄alkyl; each X is independently —I; and, the group —N(CH₂CH₂X)₂ is independently attached at the 4-position.
 19. A compound according to claim 1, wherein R^(N) is independently -Et or -Me; each X is independently —I; and, the group —N(CH₂CH₂X)₂ is independently attached at the 4-position.
 20. A compound according to claim 1, wherein R^(N) is independently -Me; each X is independently —I; and, the group —N(CH₂CH₂X)₂ is independently attached at the 4-position.
 21. A compound according to claim 1, wherein n is 0, 1, or
 2. 22. A compound according to claim 16, wherein n is
 0. 23. A compound according to claim 1, wherein each R^(P), if present, is independently halo, C₁₋₄alkyl, nitro, or cyano.
 24. A compound according to claim 1, wherein each R^(P), if present, is independently: —F, —Cl, —Br, —I, -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu, —NO₂, or —CN.
 25. A compound according to claim 1, wherein each R^(P), if present, is independently —F, —Cl, —Br, or —I.
 26. A compound according to claim 1, wherein m is 0, 1, or
 2. 27. A compound according to claim 16, wherein m is
 0. 28. A compound according to claim 22, wherein m is
 0. 29. A compound according to claim 1, wherein each R^(M), if present, is independently selected from: C₁₋₄alkyl; C₁₋₄alkoxy; amino; halo; C₁₋₄alkylthio; acyl; ester; amido; cyano; nitro; and, C₅₋₆aryl.
 30. A compound according to claim 1, wherein each R^(M), if present, is independently selected from: -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu; —CF₃, —CH₂F, —CH₂CF₃, —CH₂CH₂F; —CF₂CF₃; —OMe, —OEt, —O-nPr, —O-iPr, —O-nBu, —O-sBu, —O-iBu, —O-tBu; —OCF₃, —OCH₂F, —OCH₂CF₃, —OCH₂CH₂F; —OCF₂CF₃; —NH₂, —NMe₂, —NEt₂, —N(nPr)₂, —N(iPr)₂, —F, —Cl, —Br, —I; —SMe, —SEt; —C(═O)Me; —C(═O)OMe, —C(═O)OEt; —CONH₂, —CONHMe; —CN; —NO₂; and, -Ph.
 31. A compound according to claim 1, wherein each R^(M), if present, is independently selected from: -Me, -Et, —CF₃, —OMe, —OEt, —NH₂, and —NMe₂.
 32. A compound according to claim 1, wherein each R^(G) is independently —H.
 33. A compound according to claim 1, wherein each R^(G) is independently —H, unsubstituted C₁₋₇alkyl, substituted C₁₋₇alkyl, or silyl.
 34. A compound according to claim 1, wherein each R^(G) is independently —H; unsubstituted C₁₋₄alkyl; C₁₋₄alkyl substituted with one or more groups selected from optionally substituted C₅₋₂₀aryl, C₁₋₇alkoxy, C₁₋₇alkylthio, and acyloxy; or —SiR^(S) ₃, wherein each R^(S) is independently —H or C₁₋₄alkyl.
 35. A compound according to claim 1, wherein each R^(G) is independently —H; -Me; -Et; -nPr; -iPr; -allyl; -nBu; -sBu; -iBu; -tBu; C₁₋₄alkyl substituted with one or more groups selected from optionally substituted phenyl, methoxy, methylthio, acetoxy, and benzoyloxy; —Si(Me)₃; —Si(Et)₃; —Si(iPr)₃; —Si(tBu)(CH₃)₂; or —Si(tBu)₃.
 36. A compound according to claim 1, wherein each R^(G) is independently (1) t-butyl, (2) allyl, (3) tri-isopropylsilyl, (4) acetoxymethyl, (5) methoxymethyl, (6) methylthiomethyl, (7) p-methoxyphenylmethyl, (8) bis(o-nitrophenyl)methyl, (9) benzyl, or (10) diphenylmethyl.
 37. A compound according to claim 1, wherein each R^(G) is independently (1) t-butyl, (2) allyl, or (3) tri-isopropylsilyl.
 38. A compound according to claim 1, wherein each R^(G) is independently (1) allyl.
 39. A compound selected from compounds of the following formula (P-1), and pharmaceutically acceptable salts, solvates, amides, and esters thereof:


40. A compound selected from compounds of the following formula (P-2), and pharmaceutically acceptable salts, solvates, amides, and esters thereof:


41. A compound selected from compounds of the following formula (P-3), and pharmaceutically acceptable salts, solvates, amides, and esters thereof:


42. A composition comprising a compound according to claim 1, and a pharmaceutically acceptable carrier.
 43. A kit comprising: (a) a compound according to claim 1; and (b) instructions for use.
 44. A kit comprising: (a) a compound according to claim 1; (b) an antibody or fragment thereof conjugated or fused to a carboxypeptidase enzyme; and, (c) instructions for use.
 45. A kit comprising: (a) a compound according to claim 1; (b) a nucleic acid encoding a carboxypeptidase enzyme; and, (c) instructions for use.
 46. A method of inhibiting cell cycle progression of a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound according to claim
 1. 47. A method of treatment of colon cancer comprising administering to a subject in need of treatment a therapeutically-effective amount of a compound according to claim
 1. 48. A method of inhibiting cell cycle progression of a cell, in vitro or in vivo, comprising contacting the cell with a therapeutically-effective amount of a compound according to claim 1, in the presence of a carboxypeptidase enzyme.
 49. A method of treatment of colon cancer comprising administering to a subject in need of treatment a therapeutically-effective amount of a compound according to claim 1, in the presence of a carboxypeptidase enzyme.
 50. A method of inhibiting cell cycle progression of a cell, in vitro or in vivo, comprising: (i) contacting the cell with an antibody or fragment thereof conjugated or fused to a carboxypeptidase enzyme; and, (ii) contacting the cell with a therapeutically-effective amount of a compound according to claim
 1. 51. A method of treatment of colon cancer, comprising administering to a subject in need of treatment: (i) an antibody or fragment thereof conjugated or fused to a carboxypeptidase enzyme; and, (ii) contacting the cell with a therapeutically-effective amount of a compound according to claim
 1. 52. A method of inhibiting cell cycle progression of a cell, in vitro or in vivo, comprising: (i) contacting the cell with a nucleic acid encoding a carboxypeptidase enzyme; and, (ii) contacting the cell with a therapeutically-effective amount of a compound according to claim
 1. 53. A method of treatment of colon cancer, comprising administering to a subject in need of treatment: (i) a nucleic acid encoding a carboxypeptidase enzyme; and, (ii) a therapeutically-effective amount of a compound according to claim
 1. 